* 


LIBRARY 

OP  THE 

UNIVERSITY  OF  CALIFORNIA. 

81OUOGT 

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MEDIUM    CONTINENTAL,  THREE-FIFTHS    ACTUAL    SIZE. 

Bausch  and  Lomb  Optical  Company. 

A,  base  or  foot;  B,  pillar;  C,  arm,  in  it  the  spring  for  the  fine  adjustment;  D,  body; 
E,  objective;  F,  draw-tube;  G,  eye-piece  or  ocular;  H,  mirror;  I,  sub-stage  condenser;  J, 
stage;  K,  slide;  L,  fine  adjustment;  M,  coarse  adjustment. 


DIRECTIONS 


FOR  WORK  IN  THE 


H1STOLOGICAL  LABORATORY 


MORE   ESPECIALLY  ARRANGED  FOR  THE  USE  OF 
CLASSES  IN  THE  UNIVERSITY  OF  MICHIGAN 


G.  CARL  HUBER,  M.  D. 
n 

Assistant  Professor  of  Histology  and  Embryology 


SECOND    EDITION. 


GEORGE  WAHR 

PUBLISHER  AND  BOOKSELLER 
ANN  ARBOR,  MICHIGAN 


H 


COPYRIGHTED 
GEO.  WAHR 

IS95 


THE  COURIER  PRESS 
ANN  AKBOR 


PREFACE  TO  THE  SECOND  EDITION. 

The  subject  matter  of  the  first  part  of  these  laboratory  notes  has  been 
almost  entirely  rewritten;  the  lessons  have  been  rearranged;  some  few  of  the 
exercises  outlined  in  the  first  edition,  have  been  replaced  by  others,  and 
.many  new  ones  added.  The  course,  as  now  outlined,  covers  the  essentials 
of  normal  histology. 

The  second  part  has  been  amplified,  and  many  new  methods  added. 

I  wish  to  thank  my  assistant,  Dr.  Alfred  B.  Olsen,  for  aid  rendered  in 
proof  reading  and  other  assistance  given. 

Ann  Arbor,  June,  1895. 

G.  CARL  HUBER. 


PREFACE  TO  THE  FIRST  EDITION. 

The  following  pages  have  been  prepared  with  the  hope  that  they  may 
serve  as  a  guide  and  a  help  to  the  students  doing  work  in  the  Histological 
Laboratory.  In  the  very  brief  description  that  is  given  of  the  tissues  to  be 
studied,  I  have  born  in  mind  that  the  students  entering  on  this  work  will 
have  had  a  course  of  lectures  on  Histology.  The  aim  of  the  notes  is  there- 
fore not  to  supplant,  but  rather  to  supplement  the  text-books  on  this  subject. 

Drawings  are  to  be  made  of  nearly  all  the  preparations  examined. 
There  is  no  better  way  for  the  student  to  obtain  a  correct  and  a  lasting 
impression  of  the  tissues  to  be  studied  than  by  carefully  sketching  what  he 
sees  under  the  microscope.  The  drawings  are  to  be  made  in  the  laboratory. 
A  few  methods  for  hardening,  macerating,  embedding,  and  staining  tissues 
are  given;  such  as  have  proved  themselves  to  be  most  reliable  have  been 
selected.  Anyone  familiar  with  the  methods  here  given  can  without  diffi- 
culty employ  any  he  may  find  recorded  in  the  works  on  microscopical  technic. 

I  am  indebted  to  the  Bausch  &  Lomb  Optical  Company  for  sectional 
diagram  of  their  Model  microscope;  to  Mr.  S.  P.. Budgett  for  the  drawings 
of  the  two  wood  cuts.  I  also  wish  to  acknowledge  the  help  received  in  the 
arrangement  of  lessons,  from  Schaffer's  Essentials  of  Histology. 

G.  C.  H. 


198471 


SUPPLIES. 

The  student  before  entering  on  his  work  should  supply  himself  with 
the  following  outfit: — 

One  gross  of  slides. 

Cover  glasses.     One  oz.  of  No.  2  #  inch  squares. 

Two  dozen  No.  2  fy  inch  circles. 

One  dozen  No.  2    I  in.  by  ^  or  ^  inch. 
Slide  boxes. 

Drawing  pencil  and  blender.     Two  droppers.     Two  teasing    needles. 
Section  lifter.     Two  camel's-hair  brushes. 
Four  solid  watch  crystals.     A  pair  of  cover  glass  forceps. 
A  section  knife  (a  razor  flat  on  one  side  will  answer). 
A  tube  of  Canada  balsam.     Slide  labels. 


BOOKS  OF  REFERENCE. 

HISTOLOGY. 

Piersol. — Normal  Histology. 

Toldt. — Lehrbuch  der  Gewebelehre. 

Behrens,  Kossel,  Schiefferdecker. — Die  Gewebe  des  Menschlichen 
Korpers. 

Kolliker. — Handbuch  der  Gewebelehre  des  Menschen. 

Schwalbe. — Lehrbuch  der  Anatomic  der  Sinnesorgane. 

Cadiat. — Traite"  d'Anatomie  Generate. 

Quain. — Elements  of  Anatomy.  Vol.  I.  Part  II.  General  Anatomy 
or  Histology,  by  Prof.  Schaffer. 

Orth.— Normale  Histologie. 

Stdhr. — Lehrbuch  der  Histologie. 

MICROSCOPICAL  TECHNIC. 

Lee. — The  Microtomist's  Vade-Mecum. 

Whitman. — Methods  of  Microscopical  Anatomy  and  Embryology. 

Behrens. — Tabellen  zum  Gebrauch  bei  mikroskopischen  Arbeiten. 

Ranvier. — Traite  technique  d'Histologie. 

Boehm  and  Oppel. — Taschenbuch  der  mikroskopischen  Technik. 

Friedlander-Eberth. — Mikroskopische  Technik  zum  Gebrauch  bei 
Medicinischen  und  pathologisch-anatomischen  Untersuchungen. 

Kahlden. — -Technik  der  histologischen  Untersuchung  pathologisch- 
anatomischer  Priiparate. 

Rawitz. — Leiffaden  der  histologischen  Untersuchungen. 

Behrens,  Kossel,  Schiefferdecker. — Vol.  I. 


LESSON  I. 
CELL  AND  CELL  DIVISION. 

(a)  Plant  Cells. 

From  one  of  the  layers  of  an  onion  remove  with  forceps  a  small 
strip  of  the  film  which  covers  its  inner  surface.  Spread  out  this 
membrane  on  a  slide  in  a  drop  of  water,  and  cover  with  a  cover 
glass.  Use  the  low  power.  In  studying  an  unstained  preparation, 
it  is  best  to  employ  one  of  the  smaller  openings  in  the  diaphragm, 
thus  cutting  off  all  unnecessary  light. 

Observe  the  large  cells,  oblong  or  nearly  rectangular 
in  shape,  each  surrounded  by  a  distinct  cell  membrane. 
Notice  the  round  or  oval  nucleus  with  one  or  more  nucleoli. 
Carefully  elevate  the  cover  glass  at  one  edge,  and  add  a 
drop  or  two  of  Lugol's  solution  or  tincture  of  iodine,  and 
again  replace  the  cover  slip.  In  a  few  moments  the  cells 
will  assume  a  yellowish-brown  color.  Their  structure  can 
now  be  made  out  more  easily.  Sketch  a  number  of  the 
cells  as  seen  under  both  low  and  high  power,  after  they 
have  been  stained. 

(b)  Karyokinesis  in  Plant  Cells. 

Young  and  growing  onion  roots  were  hardened  in  Fleming's 
solution,  embedded  in  paraffin,  longitudinal  sections  cut  and  fixed 
to  cover  glasses,  and  stained  in  safranin.  Come  .to  the  table  with  a 
small  drop  of  Canada  balsam  on  your  slide,  and  the  section  will  be 
mounted  for  you. 

Under  the  low  power  it  will  be  noticed  that  some  of 
the  nuclei  are  stained  deeper  than  others.  Examine  these 
under  high  power.  Usually  they  will  show  some  one  of 
the  several  stages  of  mitotic  cell  division.  Observe  the 
difference  in  structure  between  a  resting  nucleus  and  one 
in  the  spirem  stage.  In  the  former  a  chromatic  network 
will  be  seen,  in  the  latter  the  chromosoma  can  be  clearly 


made  out.  Search  for  cells  showing  the  nucleus  in  the 
monaster,  metakinetic,  and  diaster  stages.  Sketch  a  rest- 
ing nucleus,  and  the  various  stages  of  division  seen  in 
your  section. 

(c)  Large  Animal  Cells. 

The  ovary  of  a  very  young  chicken  was  hardened  in  a  saturated 
aqueous  solution  of  bichloride  of  mercury,  stained  en  masse  in  borax 
carmine,  embedded  in  paraffin,  and  sectioned. 

All  paraffin  sections  are  to  be  mounted  in  the  following 
way : — Bring  to  the  distributing  table  a  slide  on  which  a 
thin  layer  of  albumen  fixative  (equal  parts  of  white  of 
egg  and  glycerine)  has  been  spread.  The  paraffin  section 
will  then  be  fixed  to  the  slide.  Then  hold  the  slide  over  a 
gas  flame  until  the  paraffin  begins  to  melt,  next  drop  a  few 
drops  of  turpentine  on  the  section,  and  at  the  same  time 
rock  the  slide  backward  and  forward  until  the  paraffin  has 
been  dissolved.  Wipe  away  the  excess  of  turpentine,  add 
a  small  drop  of  balsam,  and  cover  with  a  cover  glass. 

Study  under  low  power.  Observe  the  large  round  cells, 
some  of  which  show  a  relatively  large  nucleus.  Sketch 
several  as  seen  under  low  power. 

(d)  Karyokinesis  in  Animal  Cells. 

The  testes  of  a  salamander  are  to  be  removed  from  the  animal 
during  the  month  of  June.  They  were  cut  into  small  pieces,  hardened 
in  Fleming's  or  Hermann's  solution,  embedded  in  paraffin,  very  thin 
sections  cut  and  fixed  to  cover  glasses,  and  stained  either  in  safranin 
and  licht  griin  01  Haidenhain's  iron-lack-hsematoxylin  and  acid 
fuchsin.  After  staining,  the  sections  were  dehydrated,  passed  through 
oil  of  bergamot  into  xylol,  from  which  they  are  mounted.  Come  to 
the  table  with  a  small  drop  of  balsam  on  your  slide. 

Study  the  section  first  under  low  power,  and  you  will 
observe  that  the  cells  are  arranged  in  larger  and  smaller 
nests.  Under  high  power  groups  of  cells  in  mitotic  cell  , 
division  are  often  met  with.  All  the  cells  in  such  a  group 
usually  present  about  the  same  stage  of  division.  Search 
the  section  thoroughly  until  all  the  stages  have  been  seen 
and  sketched. 


(e)  Cells  Showing  Nucleus  and  Accessory  Nucle- 
us (Nebenkern). 

Small  pieces  of  a  guinea  pig's  testis  were  hardened  in  the 
following  mixture  : 

Osmic  acid,  2%  solution  2  parts. 

Platinum  chloride,  2%  solution  3     " 

Mercuric  chloride,  saturated  aqueous  sol.  5     " 

They  are  hardened  for  24  hours.*  embedded  in  paraffin,  sectioned 
and  fixed  to  cover  glasses,  stained  in  iron-lack-hsematoxylin 
(Haidenhain's),  dehydrated,  cleared  in  oil  of  bergamot,  and  passed 
into  xylol.  Come  to  the  table  with  a  small  drop  of  balsam  on  your 
slide. 

Search  for  tubules  showing  the  last  stages  of  the  devel- 
opment of  the  spermatozoa.  In  the  cells  forming  the  walls 
of  the  seminiferous  tubules,  the  accessory  nucleus  is  stained 
deeply  black,  the  nucleus  a  bluish-gray  color.  Sketch 
several  of  the  cells  showing  this  structure. 

DRAWINGS  FOR  LESSON  I. 


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DRAWINGS  FOE  LESSON  I. 


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LESSON  II. 
BLOOD. 

(a)  Fresh  Human  Blood. 

Obtain  a  drop  of  blood  by  pricking  a  carefully  cleaned  finger 
with  a  steel  pen,  one  of  the  prongs  of  which  has  been  broken  off; 
quickly  mount  the  drop  on  a  slide,  and  examine  with  the  high 
power. 

Observe  that  most  of  the  red  cells  are  arranged  in 
rouleaux,  and  between  these  now  and  then  a  white  cor- 
puscle is  seen.  If  your  drop  was  small,  some  of  the  red 
corpuscles  found  in  the  peripheral  part  of  the  preparation 
will  appear  crenated. 

(b)  Fresh  Human  Blood  with  Normal  Salt. 

Obtain  a  very  small  drop  of  blood  as  above  directed,  mix  it  on 
the  slide  with  a  drop  of  normal  salt  solution,  and  cover. 

The  smaller  number  of  corpuscles  will  allow  of  a  more 
careful  study  of  their  size  and  shape.  Observe  that  the 
red  appear  as  biconcave  circular  discs,  and  are  a  little 
smaller  than  the  majority  of  the  white.  Some  of  the  red 
will  soon  be  crenated.  Make  a  drawing  of  a  number  of 
the  red  cells  as  seen  on  u  the  flat,"  and  a  few  seen  on  the 
edge  (profile). 

(c)  Human  Blood  Stained  in  Haematoxylin  and 

Eosin. 

A  small  drop  of  blood  was  spread  between  two  clean  square 
cover  glasses  (No.  1,  %  in.);  the  cover  glasses  were  then  quickly 
drawn  apart,  and  the  thin  film  of  blood  allowed  to  dry.  The  cover 
glass  with  the  blood  was  then  placed  for  1  to  24  hours  in  a  solution 
composed  of  3  parts  of  ether  and  1  of  absolute  alcohol  which  fixes  the 
blood,  it  was  next  stained  for  15  to  20  minutes  in  Boehmer's  hsem- 
atoxylin,  and  for  5  minutes  in  a  \%  solution  of  eosin,  washed  in 
flowing  water  and  dried  between  filter  paper.  Mount  on  a  small 
drop  of  balsam. 


All  nuclei  are  stained  with  the  hsematoxylin,  the 
protoplasm  with  the  eosin.  Eosinophile  granules  are  also 
stained  red ;  the  other  granules  are  not  stained. 

On  examining  with  high  power  the  following  varieties 
of  cells  will  be  seen  in  the  preparation : 

(1)  Red  blood  cells  stained  in  eosin. 

(2)  Small  lymphocytes,  in  size  a  little  larger  than  the 
red  blood  corpuscles,  with  a  relatively  large  nucleus  stained 
quite  deeply  by  the    haematoxylin,   and   only   a   narrow 
border  of  protoplasm. 

(3)  Large  mononuclear  cells  showing  a  much  larger 
border  of  protoplasm,  and  a  nucleus  which  usually  stains 
quite  faintly. 

(4)  Transitional  form  with   a   horseshoe-shaped   nu- 
cleus.    These  develop  from  the  large  mononuclear. 

(5)  Polynuclear  cells,  with  a  lobulated  nucleus,  which 
usually  stains  quite  deeply.      They  develop  from  the  tran- 
sitional forms,  and  form-  about  70  to  75   %  of  the  white 
blood  cells. 

(6)  Eosinophile  polynuclear  cells,  with  lobulated  nu- 
cleus, and  eosinophile  granules  in  the  protoplasm. 

Make  a  sketch  of  each  of  the  above  varieties. 

(d)  Human  Blood  Stained  in  Ehrlich's  Neutrophile 

Stain. 

Blood  was  spread  and  fixed  as  in  (c)  of  this  lesson,  was  then 
stained  in  Ehrlieh's  neutrophile  stain  for  half  an  hour,  washed  in 
flowing  water,  dried  between  filter  paper,  and  ar.e  to  he  mounted 
on  balsam. 

The  red  blood  corpuscles  are  stained  a  reddish-brown, 
all  nuclei  green,  neutrophile  granules  in  the  protoplasm 
of  polynuclear  cells  a  reddish-purple,  and  eosinophile 
granules  red.  Study  under  high  power,  and  the  corpuscles 
named  in  (c)  will  be  seen.  Look  carefully  for  the  neutro- 
phile granules  in  polynuclear  cells.  They  are  not  found  in 
lymphocytes  nor  in  the  mononuclear  cells,  and  only  in  the 
latter  stages  of  the  transitional  forms. 

(e)  Amphibian  Blood    Stained  in  Hsematoxylin 

and  Eosin. 

The  heart  of  a  frog  was  opened,  and  a  small  drop  of  blood 
placed  on  a  cover  glass,  quickly  covered  with  another  cover  a;lass, 


—  7  — 

and  the  two  covers  drawn  apart,  next  fixed  in  ether  and  alcohol, 
stained  in  luematoxylin  and  eosin,  washed  and  dried.  Mount  in 
balsam. 

Study  under  high  power.  The  red  blood  corpuscles 
are  oval,  somewhat  flattened  and  nucleated.  When  seen 
in  profile  they  show  a  slight  convexity  in  the  center.  Ob- 
serve the  white  corpuscles  which  are  smaller  than  the  red. 
Sketch  several  red,  and  the  several  forms  of  the  white  met 
with. 

('f )  Pigeon  Blood  Stained  in  Hsematoxylin  and 
Eosin. 

One  of  the  toes  of  a  pigeon  was  amputated.  From  the  flowing 
blood  a  small  drop  was  caught  on  the  cover  glass,  spread,  fixed,  and 
stained  in  htematoxylin  and  eosin.  Mount  in  balsam. 

The  red  cells  are  oval,  somewhat  flattened,  and  nucle- 
ated. Observe  the  white  cells.  Sketch  red  and  white  cor- 
puscles. 

(g)  Amoeboid  Movement  in  White  Blood  Cells  of 
a  Frog. 

A  small  quantity  of  blood  obtained  from  the  heart  of  a  frog  is 
mixed  with  a  drop  of  normal  salt  solution  and  covered  with  a  slip. 
To  prevent  evaporation  while  examining,  the  cover  glass  is  fixed  by 
means  of  two  or  three  drops  of  melted  paraffin,  so  placed,  that  after 
congealing,  one-half  of  the  drop  will  rest  on  the  slide,  the  other  on 
the  edge  of  the  cover;  the  sealing  is  then  completed  by  means  of  a 
camel's-hair  brush  which  has  been  dipped  in  vaseline  and  drawn 
about  the  edge  of  the  cover. 

In  examining  this  preparation  be  very  careful  not  to 
bring  the  end  of  the  objective  in  contact  with  the  vaseline 
on  the  slide.  Search  the  specimen  until  you  find  a  white 
blood  cell  of  somewhat  irregular  shape ;  place  it  in  the 
center  of  the  field,  and  make  a  drawing  of  it  as  it  presents 
itself  to  you  at  this  time ;  at  intervals  of  one  or  two  min- 
utes carefully  repeat  the  sketching  until  about  ten  sketches 
have  been  made.  You  will  no  doubt  see  that  the  cell  in 
question  has  changed  its  shape  a  number  of  times,  pseu- 
dopodia  may  have  been  thrown  out  and  again  withdrawn, 
etc. 


DRAWINGS  FOR  LESSON  II. 


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LESSON  III. 

BLOOD  (Concluded). 

(a)  Fibrin. 

Place  a  large  drop  of  blood  on  the  slide,  allow  it  to  clot  par- 
tially ; cover  with  a  cover  slip.  Wash  gently  with  a  current  of  water 
until  nearly  all  the  red  blood  cells  have  been  washed  out.  Run  a 
few  drops  of  a  \%  solution  of  methylene  blue  under  the  cover 
glass. 

Examine  with  high  power.  Small  glistening  fibrillse 
woven  into  a  network  will  be  seen  ;  these  are  fibrin  filaments. 
The  nuclei  of  the  white  blood  cells  will  be  stained  blue. 

(b)  Teichmann's  Crystals,  Hsemin  Crystals,  Chlo- 

ride of  Hsematin. 

Several  weeks  ago  the  blood  of  a  mamin:il  was  allowed  to  flow 
on  a  piece  of  cloth.  Cut  out  a  small  piece  of  the  cloth  with  a  por- 
tion of  the  blood-stain  on  it.  Tease  it  on  a  slide  in  a  few  drops  of 
normal  snlt,  and  set  it  aside  until  some  of  the  stain  soaks  out  into 
the  solution.  In  case  the  normal  salt  begins  to  evaporate  add  now 
and  then  a  drop  of  the  solution  in  order  to  keep  the  teased  fibres 
well  soaked.  When  some  of  the  blood  has  been  dissolved  by  the 
salt  solution,  remove  the  cloth  fibres,  and  set  the  slide  away  to  evap- 
orate. If  care  be  taken  not  to  t/ct  the  slide  too  hot,  the  evaporation 
can  be  hastened  by  holding  the  slide  over  a  gas  flame.  When  the 
evaporation  has  been  completed,  scrape  what  is  left  on  the  slide  hit  > 
a  small  mass,  and  add  one  or  t\vo  drops  of  glacial  acetic  acid,  cover 
with  a  cover  glass,  and  heat  over  a  flame  until  bubbles  form.  Con- 
tinue the  heating  till  the  acid  has  evaporated.  Run  a  drop  of  water 
under  the  cover  glass,  and  examine  for  Teichmann's  crystals.  If 
present,  the  cover  glass  may  be  removed,  dried,  and  mounted  oh 
balsam. 

Study  under  high  power.  The  crystals  are  seen  in  the 
form  of  rhombic  plates  or  rhombic  rods  of  a  brownish-red 
color. 

(c)  Teichmann's  Cyrstals  from  Pigeon's  Blood. 

Prepare  as  (b)  of  this  lesson. 


10  

The  crystals  will  be  of  the  same  form  and  size  as  those 
made  from  mammalian  blood.  Teichmann's  crystals  can 
be  obtained  from  blood  stains  no  matter  how  old,  hence  their 
presence  is  often  of  value  in  medico-legal  cas"es.  Remem- 
ber that  the  fact  that  Teichmann's  crystals  can  be  obtained 
from  a  stain  only  shows  the  presence  of  blood  and  nothing 
more ;  they  can  be  obtained  from  the  haemoglobin  of  any 
red  blood  cell. 

(d)  Detection  of  Red   Blood   Cells  in  a  Dry  Clot 

of  Mammalian  Blood. 

Take  a  small  portion  of  a  dry  clot,  place  it.  on  a  slide,  and 
cover  it  with  several  drops  of  a  33%  solution  of  potassium  hydrate. 
After  the  clot  has  become  somewhat  softened,  break  it  up  into  as 
small  pieces  as  possible  with  your  teasing  needles,  and  cover  with 
a  cover  glass.  Set  it  aside  for  5  to  10  iniuutes,  then  gently  tap  the 
cover  glass  with  a  teasing  needle. 

Examine  to  see  whether  any  of  the  corpuscles  are  free; 
if  so,  determine  their  shape  and  size;  if  not  set  aside 
awhile  and  try  again. 

(e)  Detection  of  Red  Blood  Corpuscles   in  a  Dry 

Clot  of  Pigeon's  Blood. 

Prepare  as  (d)  of  this  lesson. 

Search  for  the  oval  red  blood  cells;  you  may  be  able 
to  make  out  a  nucleus  in  them. 


DRAWINGS  FOR  LESSOR  III. 


DRAWINGS  FOR  LESSON 


t 

LESSON  IV. 
EPITHELIUM. 

(a)  Pavement  Epithelium,  Isolated  Cells. 

Scrape  the  inside  of  the  cheek  with  a  clean  slide  or  the  edge 
of  a  knife,  mount  in  a  drop  of  saliva,  and  cover  with  a  slip. 

Study  under  higli  power,  and  make  use  of  one  of  the 
smaller  openings  in  the  diaphragm.  Observe  the  large  flat 
cells  with  oval  or  rounded  nuclei  showing  now  and  then  a 
few  granules  in  the  protoplasm.  You  will  also  see  the  sali- 
vary corpuscles,  leucocytes  which  have  wandered  through 
the  oral  epithelium.  They  are  slightly  swollen  by  the  im- 
bibition of  water. 

(b)  Isolated  Pavement  Epithelial  cells  Stained  in 

Hsematoxylin  and  Eosin. 

Tease  some  scrapings  from  the  inside  of  the  cheek  on  a  cover 
glass,  dry  the  preparation  (after  teasing)  over  the  flame,  stain  for 
15  minutes  in  Boehmer's  hsematoxylin,  wash  in  flowing  water, 
stain  in  eosin  for  5  minutes,  again  wash  in  water,  and  dry  the  prep- 
aration between  two  filter  papers.  Mount  in  balsam. 

The  nuclei  are  stained  blue,  the  protoplasm  red. 
Sketch  a  number  of  the  epithelial  cells  and  salivary  cor- 
puscles as  seen  under  the  high  power. 

(c)  Cutaneous  Epithelium  from  a  Frog's  Epider- 

mis. 

If  a  frog  is  imprisoned  for  some  time  in  a  jar  containing  a 
small  quantity  of  water,  portions  of  the  epidermis  are  from  time  to 
time  shed,  and  are  found  floating  in  the  water,  These  thin  mem- 
branes were  washed,  stained  in  Boehmer's  hsematoxyliu,  dehy- 
drated in  alcohol,  and  are  now  in  oil  of  cloves.  To  mount,  place 
the  tissue  on  a  clean  slide,  remove  excess  of  oil,  add  a  drop  of 
Canada  balsam,  arid  cover  with  a  slip. 


—  I4  — 

This  preparation  gives  you  a  surface  view  of  stratified 
pavement  epithelium.  Observe  the  flat  nucleated  cells, 
cemented  together  by  a  small  amount  of  intercellular 
cement  substance.  Sketch  a  small  portion  of  the  field  as 
seen  under  the  high  power. 

(d)  Cross  Section  of  Stratified  Pavement  Epithe- 

lium. ^ 

A  small  portion  of  the  soft  palate  or  the  oesophagus  was 
hardened  in  a  saturated  aqueous  solution  of  mercuric  chloride  or 
absolute  alcohol,  stained  in  borax  or  alum  carmine,  embedded  in 
paraffin,  and  sectioned.  Mount  in  balsam. 

Observe  the  several  layers  of  epithelial  cells,  and  their 
change  in  shape  as  they  are  traced  outwards.  In  the  low- 
est layer,  the  cells  are  columnar,  in  the  following  polygonal, 
while  in  the  outermost  layers  they  are  flattened.  Notice 
the  change  in  the  shape  and  structure  of  the  nuclei  of  the 
cells  of  the  different  layers.  Sketch  as  seen  under  high 
power. 

(e)  Isolated  Transitional  Epithelial  Cells  Stained 

in  Haematoxylin  and  Eosin. 

The  bladder  of  a  large  dog  was  distended  with  33%  alcohol 
before  removing,  the  urethra  was  tied.  The  bladder  was  then  placed 
in  33%  alcohol,  where  it  remained  24  hours.  It  was  then  cut  into 
small  pieces.  Scrape  the  epithelial  surface  of  one  of  the  small 
pieces,  place  the  scraping  on  a  cover  glass,  and  tease  thoroughly. 
After  teasing  dry  the  preparation  over  a  flame.  Stain  in  hsema- 
toxylin  and  eosin,  and  mount  on  balsam. 

Examine  under  high  power.  Cells  of  various  shapes 
will  be  seen.  Some  are  large  flat  scales^with  one  or  two 
nuclei ;  others  are  pear-shaped,  spindle-shaped,  or  triangu- 
lar. The  pelvis  of  the  kidney,  the  ureter,  and  the  bladder, 
are  lined  with  transitional  epithelium.  Sketch  the  several 
forms  of  epithelial  cells  found. 

(f)  Cross  Section  of  Transitional  Epithelium. 

Small  pieces  of  the  bladder  of  a  dog  were  hardened  in  abso- 
lute alcohol,  stained  for  24  hours  in  Delafield's  hsematoxylin  (one 
part  of  the  stain  to  ten  parts  of  distilled  water),  embedded  in 
paraffin,  and  sectioned.  Fix  to  the  slide  with  albumen  fixative, 
and  mount  in  balsam. 


Study  under  high  power.  The  cells  of  the  upper 
layer  (free  border)  are  large  flat  scales  with  a  smooth  free 
edge.  The  lower  border  is  often  slightly  pitted,  these 
depressions  receiving  the  cells  of  the  next  lower  layer. 
These  large-cells  have  one  or  two  nuclei.  The  cells  of  the 
second  layer  are  pear-shaped  or  irregularly  columnar.  The 
cells  of  the  deeper  layer  are  triangular  or  spindle-shaped, 
and  fit  in  between  the  cells  of  the  second  layer.  Sketch  a 
portion  of  the  epithelium. 

DRAWINGS  FOR  LESSON  IV. 


*» 


¥*¥ 

' 


DRAWINGS  FOR  LESSON  ffV 


re 


LESSON  V. 
EPITHELIUM  (Concluded). 

(a)  Isolated  Columnar  Epithelial  Cells. 

The  small  intestine  of  a  cat  was  opened,  and  macerated  for  24 
to  48  hours  in  33%  alcohol.  Tease  a  small  portion  of  the  mucous 
membrane  in  gum  glycerine. 

Study  under  high  power,  and  use  one  of  the  smaller 
openings  in  the  diaphragm.  Observe  the  shape  of  the 
cells,  position  and  shape  of  nucleus,  and  the  striated 
border.  Sketch  as  seen  under  high  power. 

(b)  Isolated   Columnar    Cells  Stained  in  Heema- 

toxylin. 

Tease  very  thoroughly  a  small  portion  of  the  above  on  a 
cover  glass.  Dry  over  the  flame,  stain  in  hsematoxylin,  and  mount 
on  balsam. 

Observe  the  columnar  cells,  the  faintly  striated  proto- 
plasm, the  cuticular  border,  and  the  structure  of  the 
nucleus.  Sketch  a  few  of  the  cells  as  seen  under  high 
power. 

(c)  Cross  Section  of  Simple  Columnar  Epithelium. 

The  small  intestine  of  a  cat  was  cut  open,  pinned  out  on  a 
cork,  hardened  in  absolute  alcohol,  stained  in  Delafield's  h^ema- 
toxylin,  and  embedded  in  paraffin.  Cross  sections  of  the  villi  were 
made.  Fix  to  slide  and  mount  in  balsam. 

Under  low  power  you  will  observe  many  small,  nearly 
round  or  oval  patches  of  tissue.  When  one  of  these  is 
studied  under  high  power,  it  will  be  found  to  be  surrounded 
by  a  single  layer  of  columnar  cells  cemented  together  by  a 
small  amount  of  intercellular  cement  substance.  In  the 
intercellular  cement  a  leucocyte  is  now  and  then  found. 

Sketch  as  seen  under  high  power. 
2 


—  i8  — 

(d) '  Goblet    Cells    Stained  in  Haematoxylin  and 
Eosin. 

The  large  intestine  of  a  cat  was  macerated  in  33%"  alcohol  24  to 
48  hours.  Tease  a  small  portion  of  the  epithelium  on  a  cover  glass, 
dry  over  the  flame,  and  stain  in  hgematoxylin  and  eosiu.  Mount 
in  balsam. 

Study  under  high  power.  Some  of  the  cells  observed 
are  ordinary  columnar  cells;  others  are  distended  with 
mucinogen,  the  nucleus  being  pressed  down  into  the  lower 
part  of  the  cell ;  these  are  the  goblet  cells.  Sketch  several 
as  seen  under  high  power. 

(e)  Isolated  Ciliated    Columnar  Cells  Stained  in 

Hsematoxylin  and  Eosin. 

The  trachea  of  a  cat  was  macerated  in  33%  alcohol.  Tease  a 
portion  of  the  epithelium  on  a  cover  glass,  and  stain  in  hcema- 
toxylin  and  eosin.  Mount  in  balsam. 

Study  under  high  power.  Observe  that  the  majority 
of  the  cells  are  columnar  in  shape,  possessing  an  oval 
nucleus.  Notice  particularly  the  clusters  of  fine  short 
hairs  adhering  to  the  free  end  of  some  of  the  columnar 
cells.  A  few  goblet  cells  may  be  found.  Sketch  as  seen 
under  high  power. 

(f)  Cross- Section  of  Stratified  Ciliated  Columnar 

Epithelium. 

The  respiratory  mucous  membrane  of  a  dog's  nose  was  hardened 
in  mercuric  chloride,  stained  in  Delafield's  hsematoxylin,  embedded 
in  paraffin,  and  sectioned.  Mount  in  balsam. 

Study  under  high  power.  This  epithelium  is  com- 
posed of  three  layers  of  cells,  the  free  cells  being  ciliated. 
Sketch  a  portion  of  epithelial  border. 


DRAWINGS  FOR  LESSON  V. 


DRAWINGS  FOR  LESSON 


M 


21 


LESSON  VI. 
FIBROUS  CONNECTIVE  TISSUE. 

(a)  Fibrils  of  White  Fibrous  Connective  Tissue. 

One  of  the  small  tendons  of  a  rat's  tail  is  to  be  placed  on  a 
clean  and  dry  slide.  Place  a  small  piece  of  filter  paper  (about  34  in. 
square)  moistened  in  normal  salt  over  the  middle  part  of  the  tendon  ; 
quickly  spread  out  the  ends  of  the  tendon  with  your  teasing  needles. 
The  spread-out  ends  will  dry  and  fix  the  tendon  to  the  slide.  Tease 
the  middle  portion  in  normal  salt,  and  cover  with  a  slip. 

Study  under  high  power.  Observe  the  very  fine 
fibrill se,  often  slightly  wavy.  Notice  that  they  neither 
branch  nor  anastomose.  Add  a  few  drops  of  a  1%  solu- 
tion of  acetic  acid.  This  causes  the  fibrils  to  swell  up  and 
become  homogeneous.  Sketch  as  seen  before  acetic  acid 
is  added. 

(b)  Fibrils  of  Yellow  Elastic  Connective  Tissue. 

Tease  a  small  portion  of  the  elastic  tissue  taken  from  the  liga- 
mentum  nuchse  of  an  ox  in  normal  salt  solution. 

Observe  the  fibrils  of  yellow  elastic  tissue.  They  are 
highly  refractive,  branch  and  anastomose,  and  the  broken 
ends  curl  back.  Run  a  few  drops  of  the  acetic  acid  solu- 
tion under  the  cover  glass,  and  notice  that  the  yellow 
elastic  fibrils  are  not  affected  by  it. 

(c)  Stained  Yellow  Elastic  Tissue. 

Small  pieces  of  ligamentum  nuchse  were  macerated  for  3  to  4 
days  in  a  \%  solution  of  acetic  acid,  stained  for  several  days  in  a 
\%  aqueous  solution  of  acid  fuchsin,  thoroughly  washed  in  flowing 
water,  and  placed  in  glycerine.  Tease  in  gum  gl3rcerine. 

The  stained  fibrils  will  be  easily  made  out.  Sketch 
under  high  power. 


22  

(d)  Areolar  Connective  Tissue. 

A  small  portion  taken  from  the  subcutaneous  tissue  of  a  young 
cat  is  placed  on  a  dry  slide,  the  edges  are  drawn  out  and  made  to 
adhere  to  the  slide  by  allowing  them  to  dry,  while  the  center  is  kept 
moist  by  breathing  upon  it.  In  this  way  a  thin  film  is  obtained. 
Place  a  drop  of  normal  salt  on  a  cover  glass  and  invert  it  over  the 
center  of  the  film. 

Examine  under  high  power,  using  one  of  the  smaller 
openings  of  the  diaphragm.  Observe  the  bundles  of  white 
fibrous  tissue,  composed  of  elementary  fibrils,  crossing  the 
field  in  every  direction,  and  forming  a  felted  network.  On 
and  between  these  bundles,  yellow  elastic  fibres  will  be  seen. 
They  have  a  more  definite  outline,  and  branch  and  anasto- 
mose. Search  for  connective  tissue  cells. 

Carefully  elevate  the  cover  glass,  and  add  a  drop  of  1%  solu- 
tion of  acetic  acid. 

In  a  few  moments  the  bundles  of  white  fibrous  tissue 
will  swell  and  appear  homogeneous,  while  the  unaffected 
yellow  elastic  fibres  will  stand  out  boldly ;  the  nuclei  of  the 
fixed  connective  tissue  cells  can  now  be  more  clearly  seen. 
Sketch  a  bundle  of  white  fibrous  connective  tissue,  some 
yellow  elastic  fibres,  and  a  number  of  the  connective  tissue 
cells. 

(e)  Cell  Spaces  in  Areolar  Connective  Tissue. 

A  small  piece  of  areolar  connective  tissue  was  removed  from  a 
young  animal,  spread  out  on  a  dry,  clean  slide,  immersed  for  15 
minutes  in  a  1%  solution  of  silver  nitrate,  rinsed  in  water,  placed  in 
95%  alcohol,  and  exposed  to  direct  sunlight.  The  sunlight  reduces 
the  silver  nitrate  giving  the  tissue  a  brown  color.  It  was  then 
removed  from  the  slide,  hardened  in  alcohol,  and  cleared  in  oil  of 
bergamot.  Mount  in  balsam. 

Study  under  high  power.  The  ground  substance  of 
the  tissue  is  stained  brown,  and  the  irregular,  branched, 
and  often  anastomosing  cell  spaces  are  left  unstained,  and 
thus  are  clearly  seen.  Sketch. 

(f )  Tendon. 

Tendons  taken  from  the  tail  of  a  rat  were  suspended  in  alum 
carmine  for  24  hours.  Tease  in  gum  glycerine. 


—  23  — 

The  tendon  fasciculi  are  somewhat  swollen ;  between 
them  rows  of  tendon  cells  which  have  taken  the  red  color 
will  be  distinctly  seen ;  they  are  oblong  or  square  in  shape, 
with  the  nucleus  usually  at  one  end.  Sketch  two  fasciculi 
with  the  intervening  cells. 

(g)  Cross-Section  of  Tendon. 

The  skin  covering  the  tail  of  a  rat  was  removed,  the  tail  cut  in 
small  pieces,  fixed  in  a  saturated  aqueous  solution  of  mercuric 
chloride,  washed  in  water,  hardened  in  alcohol,  decalcified  in  a  \%* 
solution  of  hydrochloric  acid,  stained  in  borax  carmine,  embedded 
in  paraffin,  and  cross-sectioned.  Fix  to  the  slide  and  mount  in 
balsam. 

--  *7    -   £'• 

Examine  first  under  low  power.  (Four  small  tendons 
will  be  seen  in  cross  section)  Place  one  of  these  in  the 
field,  and  examine  under  high  power.  Observe  that  the 
tendon  is  surrounded  by  an  areolar  connective  tissue 
sheath.  The  ends  of  the  tendon  fasciculi  are  seen  in  cross 
section.  Notice  the  tendon  corpuscles  between  the  fascic- 
uli; they  usually  have  a  triangular  body  with  long 
processes.  They  are  stained  more  deeply  than  the  fibrous 
tissue.  Sketch  as  seen  under  high  power. 

DRAWINGS  FOE  LESSON  VI. 


C*-) 


DRAWINGS  FOR  LESSON  VII 


r 


LESSON  VII. 
CONNECTIVE  TISSUE  (Concluded). 

(a)  Cross-Section  of  Ligamentum  Nuchae. 

Small  pieces  of  the  ligamentum  nuchse  of  an  ox  were  hardened 
in  picric  acid,  stained  in  borax  carmine,  embedded  in  paraffin,  and 
sectioned.  Fix  to  the  slide  and  mount. 

The  cut  ends  of  the  yellow  elastic  fibres  have  an  irreg- 
ular angular  shape.  They  are  grouped  into  larger  or 
smaller  bundles.  These  bundles  are  separated  from  each 
other  by  a  small  amount  of  white  fibrous  tissue.  Here  and 
there  a  connective  tissue  cell  between  the  yellow  elastic 
fibres  is  to  be  noticed.  Sketch  as  seen  under  high  power. 

(b)  Longitudinal  Section  of  Ligamentum  Nuchse. 

The  section  was  prepared  as  above,  and  longitudinal  sections 
made.  Fix  to  the  slide  and  mount  in  balsam. 

Observe  the  anastomosing  fibres  of  yellow  elastic  tis- 
sue. Sketch  as  seen  under  high  power. 

(c)  Embryonic  Connective  Tissue. 

The  umbilical  cord  of  a  human  foetus  was  hardened  in  a  satu- 
rated aqueous  solution  of  mercuric  chloride,  stained  in  Delufield's 
hsematoxylin,  embedded  in  paraffin,  and  sectioned.  Fix  to  the 
slide  and  mount  in  balsam. 

The  umbilical  vessels  will  be  seen  occupying  the  central 
part  of  the  section.  In  this  section,  however,  the  connec- 
tive tissue  is  to  receive  special  attention.  Observe  the- 
branched  connective  tissue  cells  with  their  long  processes. 
The  cells  are  separated  by  a  large  amount  of  a  granular, 
intercellular  ground  substance,  in  which  fine  connective 
tissue  fibrils,  as  well  as  the  highly  refractive  elastic  fibres, 
may  be  seen.  Make  a  sketch  as  seen  under  high  power. 


—  26  — 

(d)  Fat  Cells. 

The  mesentery  of  a  cat  was  spread  on  a  large  glass  slide, 
hardened  for  48  hours  in  95%  alcohol,  stained  in  hsematoxylin, 
washed  in  water,  dehydrated,  and  cleared  in  oil  of  bergamot.  A 
small  portion  including  an  arteriole  is  cut  off  with  scissors,  trans- 
ferred to  the  slide,  and  mounted  in  balsam. 

Study  first  under  low  power,  and  observe  the  large, 
clear,  round  or  oval,  cells,  arranged  in  rows  or  small  groups 
on  either  side  of  the  small  vessels.  Under  high  power  a 
nucleus  is  now  and  then  made  out.  A  network  of  capil- 
laries embracing  the  fat  cells  is  often  observed.  Sketch  as 
seen  under  low  power. 

(e)  Section  of  Adipose  Tissue. 

A  small  portion  of  the  skin  was  hardened  in  absolute  alcohol, 
stained  in  borax  carmine,  embedded  in  paraffin,  and  sectioned. 
Fix  to  the  slide  and  mount. 

Study  under  low  power.  Notice  the  groups  of  fat  cells, 
lobules  of  fat,  separated  by  bundles  of  fibrous  connective 
tissue.  Sketch  as  seen  under  low  power. 

(f)  Endothelial  Cells. 

A  \%  solution  of  silver  nitrate  was  injected  into  the  peritoneal 
cavity  of  a  frog.  After  15  minutes  the  intestines  with  the  attached 
mesentery  were  removed,  pinned  out  on  a  small  piece  of  cork,  placed 
into  95%  alcohol,  and  exposed  to  the  sunlight  until  it  assumed  a 
brown  color.  After  dehydration  the  tissue  was  cleared  in  oil  of 
bergamot.  Mount  in  balsam. 

Study  under  high  power,  and  observe  that  the  silver  is 
deposited  in  the  intercellular  cement  substance,  a  delicate 
black  line  surrounding  and  outlining  each  cell.  Now  and 
then  the  protoplasm  and  nuclei  are  stained  a  light  brown. 
Sketch  as  seen  under  high  power. 


DRAWINGS  FOR  LESSON  VII. 


DRAWINGS  FOR  LESSON  VIE 


—  29  — 


LESSON  VIII. 
CARTILAGE. 

(a)  Hyaline,  Fresh. 

Thin  sections  are  removed  from  the  articular  surface  of  a  frog's 
femur  by  a  sharp  razor.  Mount  in  normal  salt  solution. 

Examine  under  high  power,  using  one  of  the  smaller 
openings  in  the  diaphragm.  Observe  the  round  or  oval 
cartilage  cells,  each  surrounded  by  a  capsule,  and  possess- 
ing a  large  spherical  or  oval  nucleus.  The  cells  are  often 
found  in  groups  of  2,  4  or  more,  and  are  in  small  spaces 
(the  lacunae)  in  the  homogeneous,  intercellular  ground 
substance,  the  matrix.  ' 

(b)  Hyaline  Cartilage,  Stained. 

The  head  of  a  femur  of  a  cat  was  divided  into  several  pieces  ; 
these  were  hardened  in  a  saturated  aqueous  solution  of  mercuric 
chloride,  decalcified  in  a  1%  solution  of  hydrochloric  acid,  stained  in 
borax  carmine,  embedded  in  paraffin,  and  cross-sections  made. 

Study  first  under  low  power.  Observe  the  hyaline 
cartilage  surrounding  the  cancellated  bone.  In  the  deeper 
portion  of  the  cartilage,  the  matrix  is  granular;  this  is  a 
narrow  zone  of  calcified  cartilage.  Sketch  the  section  as 
seen  under  low  power. 

(Q)  Elastic  Fibro-Cartilage. 

The  epiglottis  of  a  dog  was  hardened  in  alcohol,  embedded  in 
celloidin,  stained  in  haematoxylin,  and  counter-stained  in  a  1% 
aqueous  solution  of  acid  fuchsia. 

Study  under  low  power.  The  nuclei  of  the  cartilage 
cells  are  stained  with  the  hsematoxylin.  An  interlacing 
network  of  yellow  elastic  fibres  stained  red  by  the  acid 
fuchsin  will  be  seen  in  the  ground  substance.  Sketch  as 
seen  under  high  power. 


—  3°  — 

(d)  White  Fibre-Cartilage. 

Small  pieces  of  the  intervertebral  discs  of  a  calf  were  hardened 
in  a  saturated  aqueous  solution  of  picric  acid,  washed  in  alcohol, 
stained  in  picro-carmine,  embedded  in  paraffin,  and  sectioned. 

Study  under  high  power.  The  intercellular  matrix  is 
composed  largely  of  white  fibrous  tissue  stained  yellow  in 
the  picric  acid.  Between  these  bundles  are  found  cartilage 
cells,  enclosed  in  a  capsule,  and  often  surrounded  by  a 
narrow  zone  of  hyaline  matrix.  Sketch  as  seen  under  high 
power. 

DRAWINGS  FOR  LESSON  VIII. 


DRAWINGS  FOR  LESSON  VIII. 


~ 
- 

- 


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3 


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DRAWINGS  FOE  LESSON  VIII. 


m 

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v  -      ~ 


—  33  — 
LESSON  IX. 


BONE. 

(a)  Cross-Section  of  Bone. 

From  a  thoroughly  macerated  and  dried  bone  a  thin  cross- 
section  is  removed  from  the  shaft  by  means  of  a  fine  saw.  This  is 
ground  between  two  hones  until  it  becomes  very  thin  and  trans- 
parent, care  being  taken  to  keep  the  stones  well  moistened  with 
water,  and  not  to  use  too  much  pressure.  The  section  is  now 
washed,  first  in  distilled  water,  and  then  in  alcohol.  This  is  best 
done  by  means  of  a  fine  camel's-hair  brush,  using  a  deep  watch 
crystal.  In  order  to  determine  whether  your  section  is  clean, 
mount  it  in  a  drop  of  alcohol,  and  examine  under  the  microscope. 
The  Haversian  canals  and  the  lacunae  must  be  free  from  sand,  as 
they  will  be  if  the  section  is  well  washed.  If  clean,  dry  between 
two  filter  papers.  A  dry  bone  section  must  be  mounted  in  hard 
balsam.  To  prepare  this,  place  a  large  drop  of  balsam  on  a  slide, 
and  another  on  a  cover  glass.  Heat  both  over  the  flame  for  a  few 
moments,  and  then  set  aside  to  cool.  When  ready  the  touch  of  a 
needle  will  make  a  dimple  in  the  balsam.  Then  place  the  bone 
section  on  the  balsam  and  cover  with  the  prepared  cover  slip. 
Gently  press  the  cover  with  a  needle  until  the  layers  of  balsam  fuse. 

Examine  under  low  power.  Observe  that  in  a  Haver- 
sian system  the  bone  lamellae  are  concentrically  arranged 
about  the  Haversian  canal.  Note  the  bone  lacunaa  between 
the  lamellae ;  these  communicate  with  one  another  by 
means  of  the  fine  canaliculi.  Look  for  the  interstitial  and 
circumferential  lamellas.  Sketch  several  Haversian  sys- 
tems as  seen  under  low  power. 

(b)  Longitudinal  Section  of  Bone. 

A  thin  longitudinal  section  is  made  from  the  shaft  of  a  bone  by 
means  of  a  fine  saw.  Grind  and  wash  as  above.  Dry  and  mount  in 
hard  balsam. 


—  34  — 

Study  under  low  power,  and  observe  the  anastomosing 
Haversian  canals.  The  bone  lamellae  and  lacunae  are 
arranged  parallel  to  them.  Sketch  under  low  power. 

(c)  Cross-Section  of  Decalcified  Bone. 

A  portion  of  the  shaft  of  a  small  bone  was  hardened  in  a 
saturated  aqueous  solution  of  mercuric  chloride,  decalcified  in  1% 
hydrochloric  acid,  stained  in  Delafield's  hsematoxylin,  washed  in 
acid  alcohol  for  several  hours,  dehydrated,  embedded  in  paraffin, 
and  sectioned.  Fix  to  the  slide  and  mount. 

Study  under  high  power.  Observe  the  Haversian 
canals;  each  contains  one  or  more  capillaries  or  small 
arterioles  in  section.  The  lamellae  are  arranged  around 
the  canals  concentrically ;  between  these  are  the  lacunae ; 
and  in  each  lacuna  a  bone  cell.  The  canaliculi  are  not 
well  shown.  The  periosteum  is  composed  chiefly  of  fibrous 
tissue.  On  the  inner  surface  of  the  periosteum  will  be 
seen  one  or  more  layers  of  short,  irregularly  cuboidal  cells, 
the  osteoblasts.  Sketch  under  high  power. 

(d)  Longitudinal  Section  of  Decalcified  Bone. 

This  was  prepared  the  same  as  the  cross-section.  Mount  in 
balsam. 

Study  under  high  power.  The  Haversian  canals  and 
lamellae  are  seen  in  longitudinal  section;  between  two 
lamellae  is  a  row  of  lacunae  containing  bone  cells.  Sketch 
as  seen  under  high  power. 


DRAWINGS  FOR  LESSON  IX. 


-'••"•'     *-     . 


r      \ 


DRAWINGS  FOR  LESSON 


TV 


<J  Tr^V  V 


i  n^  ^\ 

h^j?>n 

H^l 


—  37  — 


LESSON  X. 

CANCELLATED  BONE,  DEVELOPING  BONE, 
AND  BONE  MARROW. 

(a)  Cancellated  Bone. 

The  head  of  a  long  bone  taken  from  a  young  animal  was 
divided  into  small  pieces,  hardened  in  mercuric  chloride,  decal- 
cified in  a  1%  solution  of  hydrochloric  acid,  stained  in  Delafield's 
hsematoxylin,  embedded  in  paraffin,  and  sectioned.  Sections  were 
fixed  to  cover  glasses,  and  counter-stained  in  eosin,  dehydrated,  and 
cleared  in  oil  of  bergamot.  Mount  in  balsam. 

Study  first  under  low  power.  Observe  the  network  of 
trabeculse.  The  spaces  bounded  by  them  are  filled  with 
marrow  cells.  Under  high  power  a  layer  of  osteoblasts  is 
often  seen  lining  the  spaces.  Fragments  of  calcified  car- 
tilage, taking  the  stain  more  deeply,  may  be  seen  in  the 
trabeculae.  Sketch  as  seen  under  high  power. 

(b)  Developing  Bone-    / 

One  of  the  developing  bones  removed  from  a  foetal  limb  was 
hardened  and  decalcified  in  picro-sulphuric  acid,  embedded  in  cel- 
loidin,  sectioned,  and  stained  in  hsematoxylin  and  eosin.  Sections 
are  now  in  oil  of  bergamot,  and  are  to  be  mounted  in  balsam. 

Study  first  under  low  power.  If  the  section  comes 
from  the  center  of  the  bone  it  will  show  the  following  areas, 
named  in  order  as  they  present  themselves  when  the  sec- 
tion is  moved  from  the  articular  surface  toward  the  middle 
of  the  shaft,  where  ossification  is  most  advanced  : — 

( 1 )  Area  of  articular  cartilage. 

(2)  Area  of  flattened  cartilage  cells,  arranged  in  rows. 

(3)  Area   of    enlarged   cartilage   cells.      Calcification 
may  have  taken  place  in  the  matrix  between  the  rows. 

(4)  Area  of  ossification.     Osseous  substance  is  being 
deposited  by  the  osteoblasts  on  the  calcified  trabecuiae  of 
the  cartilage.     Small  arterioles  and  capillaries  surrounded 


by  marrow  cells  are  found  in  the  spaces  between  the  trabec- 
ulse  (the  primary  marrow  spaces).  The}  periosteal  bone  is 
being  formed,  and  is  well  marked  off  from  the  endochon- 
dral  bone.  Sketch  under  low  power,  showing  in  your  draw- 
ing the  several  areas  mentioned  above. 

(c)  Red  Marrow. 

A  small  portion  of  red  marrow  taken  from  the  femur 
of  a  guinea  pig  was  placed  on  a  cover  glass  and  covered  by 
another  cover  glass ;  the  two  were  then  pressed  together 
and  quickly  drawn  apart.  They  were  then  hardened  for 
24  hours  in  a  solution  of  three  parts  ether  and  one  of  abso- 
lute alcohol,  and  stained  in  haematoxylin  and  eosin. 
Mount  in  balsam. 

Study  under  high  power.  Find  and  sketch  the  follow- 
ing cells : — 

(1)  Mononuclear  giant  cell. 

(2)  Polynuclear  giant  cell;  the  nucleus  may  be  lobu- 
lated,  s-shaped,  or  in  the  form  of  a  ring. 

(3)  Nucleated  red  corpuscle;    the  spherical   nucleus 
takes  the  stain  very  freely. 

(4)  Erythroblasts ;  a  little  larger  than  the  red  blood 
cells,  and  containing  none  or  very  little  haemoglobin. 

(5)  Polynuclear,  transitional,  and  mononuclear  white 
blood  cells,  and  perhaps  a  few  lymphocytes. 

(6)  Myelocytes  or  red  marrow  cells,  mononuclear  and 
a  little  larger  than  the  white  blood  cells. 


DRAWINGS  FOR  LESSON  X. 


® 


DRAWINGS  FOR  LESSON  X. 


—  41  — 


LESSON  XI. 

MUSCLE. 

(a)  Fresh  Striped  Muscle. 

Tease  a  small  shred  of  muscle  taken  from  the  leg  of  a  frog  in 
normal  salt  solution. 

Study  first  under  low  power.  Observe  the  long  cylin- 
drical fibres  showing  a  transverse  striation.  Move  the 
the  slide  about  and  you  will  find  broken  fibres,  the  broken 
ends  of  which  are  often  united  by  the  sarcolemma.  Cause 
a  few  drops  of  a  1%  solution  of  acetic  acid  to  flow  under 
the  cover  glass,  and  in  a  few  moments  the  muscle  nuclei 
will  be  seen.  Make  a  sketch  showing  the  sarcolemma; 
also  a  small  segment  of  a  muscle  fibre  showing  muscle 
nuclei  as  seen  under  high  power. 

(b)  Teased  and  Stained  Striped  Muscle. 

Small  pieces  of  a  striped  muscle  were  macerated  for  about  24 
hours  in  Sihler's  macerating  fluid,  then  transferred  to  glycerine  for 
several  hours,  and  stained  in  Sihler's  heematoxylin  for  several  days. 
Tease  carefully  in  gum  glycerine. 

Study  under  low  power.  The  cross  striations  are  very 
clearly  seen,  also  the  muscle  nuclei.  Sketch  as  seen  under 
low  power. 

(c)  Branched  Striped  Muscle  Fibres. 

The  posterior  free  end  of  a  frog's  tongue  was  macerated  for 
several  hours  in  M.  Schultz's  mixture,  and  stained  in  hsematoxylin. 
Tease  a  small  portion  in  gum  glycerine. 

When  a  voluntary  muscle  fibre  is  inserted  into  a  mu- 
cous membrane  or  the  epidermis,  the  end  so  inserted  is 
often  branched.  This  preparation,  if  you  have  teased  it 
carefully,  will  show  you  these  branched  fibres.  Sketch  un- 
der high  power. 


(d)  Cross-Section  of  Voluntary  Muscle. 

One  of  the  small  muscles  of  a  mammal  was  hardened  in  a  sat- 
urated aqueous  solution  of  mercuric  chloride,  stained  in  borax  car- 
mine, embedded  in  paraffin,  and  cross-sectioned.  Fix  to  the  slide, 
and  mount  in  balsam. 

Study  first  under  low  power.  Observe  the  endomy- 
sium  and  perimysium.  Under  high  power  note  the  struc- 
ture of  the  muscle  fibres  and  the  areas  of  Cohnheim. 
Observe  the  position  of  the  muscle  nuclei.  Sketch  under 
high  power. 

(e)  Longitudinal  Section  of  Injected  Striped  Mus- 

cle. 

The  arteries  of  one  of  the  extremities  of  a  dog  were  injected 
with  a  Berlin  blue  gelatine  mass;  the  tissue  was  then  hardened  in 
alcohol,  stained  in  alum  carmine,  embedded  in.  paraffin,  and  sec- 
tions cut  parallel  to  the  course  of  the  fibres.  Fix  to  the  slide,  and 
mount  in  balsam. 

Study  under  low  power.  The  muscle  fibres  are  stained 
with  the  alum  carmine.  Between  and  over  the  fibres  are 
tortuous  blue  threads,  the  injected  capillaries,  forming  an 
anastomosing  network.  Sketch  as  seen  under  low  power. 

(f )  Cross-Section  of  Tongue. 

The  tongue  of  a  cat  was  hardened  in  absolute  alcohol,  stained 
in  Delafield's  heematoxylin,  embedded  in  paraffin,  fixed  to  cover 
glasses,  and  counter-stained  in  eosin.  Mount  in  balsam. 

Study  first  under  low  power.  Notice  the  striped  mus- 
cle fibres  cut  transversely,  longitudinally,  and  obliquely. 
The  structure  of  the  muscle  -fibres  will  be  more  clearly 
made  out  under  high  power.  The  nuclei  are  stained  blue, 
the  muscle  substance  red.  Sketch  under  low  power. 


DRAWINGS  FOR  LESSON  XI. 


> 


DRAWINGS  FOR  LESSON  XI} 


45  — 


LESSON  XII. 
MUSCLE  (Concluded). 

(a)  Nerve-Ending  in  Voluntary  Muscle. 

Small  pieces  of  the  intercostal  muscles  of  a  snake  (the  inter- 
costal or  any  other  short  muscle  of  a  small  mammal  may  be  used) 
were  placed  for  10  to  15  minutes  in  filtere  i  lemon  juice,  then  hastily 
washed  in  distilled  water,  transferred  to  a  \%  gold  chloride  solution 
for  15  to  20  minutes,  again  washed  in  water,  and  placed  in  20%  for- 
mic acid  solution  where  they  remained  (protected  from  the  light) 

24  to  48  hours.     Tease  in  glvcerine. 

/k, 
Study  first  under  low  power,  and  search  for  a  nerve 

fibre  terminating  in  an  end  plate.  The  nerve  fibre  and 
the  branched  ending  have  a  purplish  color,  the  muscle  a 
reddish  purple.  Sketch  under  high  power. 

(b)  Isolated  Heart  Muscle  Cells. 

Small  pieces  of  the  cardiac  muscle  of  a  dog  were  macerated  in 
a  30%  solution  of  caustic  potash  for  15  minutes ;  the  maceration  was 
then  interrupted  by  placing  the  tissue  in  a  saturated  acqueous 
solution  of  potassium  acetate.  Tease  in  gum  glycerine. 

Observe  the  short  oblong  cells,  one  end  usually 
branched.  The  cells  show  a  cross  and  longitudinal  stria- 
tion,  and  possess  one,  occasionally  two,  oval  nuclei.  Draw 
several  as  seen  under  high  power. 

(c)  Section  of  Heart  Muscle. 

A  small  piece  taken  from  the  ventricle  of  a  dog's  heart  was 
hardened  in  a  saturated  aqueous  solution  of  mercuric  chloride, 
stained  in  Delafield's  haematoxylin,  embedded  in  paraffin,  and 
sectioned.  Fix  to  slide,  and  mount  in  balsam. 

Study  under  high  .power.  Note  how  the  cells  are 
cemented  into  fibres,  and  these  into  anastomosing  bundles. 
In  cross  sections  of  the  muscle  the  nuclei  are  seen  occupy- 
ing the  center  of  the  cells.  Sketch  as  seen  under  4iigh 
power. 


-  46  - 

(d)  Teased  Involuntary  Muscle. 

The  muscular  coat  of  the  small  intestine  of  a  cat  was  macerated 
for  15  minutes  in  a  30%  solution  of  caustic  potash,  then  placed  in  a 
saturated  solution  of  acetate  of  potash.  Teaso  in  gum  glycerine. 

Observe  the  long,  fusiform  or  spindle-shaped  cells 
with  rod-shaped  nuclei.  The  protoplasm  often  shows  a 
longitudinal  striation.  Sketch  several  under  high  power. 

(e)  Section  of  Involuntary  Muscle. 

The  muscular  coat  of  the  small  intestine  of  a  cat  was  hardened 
in  absolute  alcohol,  stained  in  Delafield's  hsematoxylin,  embedded 
in  paraffin,  and  sectioned.  Fix  and  mount  in  balsam. 

The  fibres  in  the  longitudinal  coat  will  appear  in  cross 
section ;  note  that  they  vary  in  size,  and  only  a  few  are 
nucleated  (many  cross  sections  can  be  made  of  a  single 
cell,  but  very  few  of  them  will  pass  through  the  nucleus). 
The  circular  layer  is  cut  in  the  directiori  of  the  longitudi- 
nal" axis  of  the  cells.  Sketch  under  high  power  a  portion 
of  both  coats. 


DRAWINGS  FOR  LESSON  XII. 


DRAWINGS  FOR  LESSON  XII. 


& 


—  49 


LESSON  XIII. 
PERIPHERAL  NERVES. 

(a)  Fresh  Medullated  Nerve. 

Tease  a  piece  from  the  sciatic  nerve  of  a,  frog  in  normal  salt 
solution.  Before  covering  arrange  the  fibres  as  straight  as  possible. 

Examine  under  high  power,  employing  one  of  the 
smaller  openings  in  the  diaphragm.  Observe  the  axis 
cylinder,  seen  as  a  light  band  passing  down  through  the 
center  of  the  fibre.  It  is  surrounded  by  a  thin  glistening 
layer  usually  of  a  light  green  color,  the  medullary  sheath; 
around  this  is  the  neurilemma.  Find  the  nodes  of  Ran- 
vier;  search  for  the  nucleus  of  an  internodal  segment.-- 
Some  of  the  fibres  may  show  the  segments  of  Lantermann. 

(b)  Medullated    Nerve    Fibres  Stained  in  Osmic 

Acid. 

The  sciatic  of  a  frog  was  fixed  for  2i  hours  in  a  1%  solution  of 
osmic  acid.  Tease  very  carefully  in  gum  glycerine. 

Examine  under  high  power.  The  medullary  sheath  is 
stained  deeply  black  by  the  osmic  acid.  The  nodes  of 
fianvier  are  very  clearly  seen  in  this  preparation. 

(c)  Medullated  Nerve  Fibres  Stained  in   Hsem- 

atoxylin. 

A  small  cerebro-spinal  nerve  was  hardened  in  a  saturated 
aqueous  solution  of  picric  acid  for  48  hours,  washed  for  several  days 
in  70%  alcohol,  teased,  and  stained  in  Boehmer's  hsematoxylin  for 
about  one  hour.  Tease  thoroughly  in  gum  glycerine. 

Study  under  high  power.     The  axis  cylinder  appears 
as  a  deeply  stained  cord  passing  through  the  center  of  the 
fibre.     The  medullary  substance  has  a  reticulated  appear- ' 
ance.     The  internodal  nuclei  are  also  stained.     Sketch  as 
seen  under  high  power. 

4 


—  5°  — 

(d)  Non-Madullated    Nerve    Fibres    Stained    in 

Haematoxylin. 

The  splanchnic  nerves  of  a  dog  were  hardened  in  pirric  acid, 
and  stained  in  Boehmer's  hrematoxylin.  Tease  in  gum  glycerine. 

Examine  under  high  power.  The  great  majority  of 
the  fibres  are  non-medullated,  i.  e.,  possess  no  medullary 
sheath,  and  hence  showing  no  nodes  of  Rauvier.  Numer- 
ous nuclei  will  be  seen,  often  giving  the  fibre  a  be  ded 
appearance.  Sketch  under  high  power. 

(e)  Cross-Section  of  a  Nerve  Trunk  Hardened  in 

Osmic  Acid. 

The  sciatic  of  a  frog  was  hardened  in  a  \%  solution  of  osmic 
acid  saturated  with  picric  acid  for  24  hours,  washed  in  70%  alco'iol 
for  2  days,  embedded  in  paraffin,  and  sectioned.  Fix  to  the  slide 
and  mount  in  balsam. 

Study  under  high  power.  The  medullary  sheath  is 
stained  black,  and  the  axis  cylinder  a  grayish-black.  Now 
and  then  fine  granules  can  be  seen  in  the  axis  cylinder ; 
these  are  the  ends  of  the  primitive  fibrillae.  Sketch  undor 
high  power. 

(f)  Cross-Section  of  a  Nerve    Trunk   Stained  in 

Anilin  Blue  and  Safranin. 

The  posterior  tibial  nerve  (human)  was  hardened  for  several 
weeks  in  Miiller's  fluid,  dehydrated,  embedded  in  paraffin,  and 
sectioned.  The  sections  were  fixed  to  cover  glasses,  stained  in 
anilin  blue  andsafranin,  dehydrated,  and  cleared  in  oil  of  bergamot 
and  xylol. 

The  axis  cylinders  are  stained  blue,  the  myelin  orange, 
and  the  nuclei  red.  Study  under  low  power.  Note  that 
the  funiculi  are  held  together  by  a  loose  connective  tissue, 
the  epineurium;  in  it  groups  of  fat  cells  and  the  blood 
vessels  of  the  nerve  trunk  are  found.  Each  funiculus  is 
surrounded  by  a  dense  connective  tissue  sheath,  the  peri- 
neurium ;  this  shows  a  lamellar  structure.  The  nerve  fibres 
in  cross  section  should  be  studied  under  high  power. 
Between  the  nerve  fibres  of  a  funiculus  a  small  amount  of 
connective  tissue  is  seen,  the  endoneurium.  Draw  under 
low  power  a  number  of  funiculi,  and  the  surrounding 


perineurium  and  epineurium;  and  under  high  power  a 
small  portion  of  a  funiculus  showing  the  fibres  in  cross 
section. 

(g)    Longitudinal    Section    of    a    Nerve    Trunk 
Stained  in  Anilin  Blue  and  Safranin. 

The  section  was  prepared  as  above,  and  the  reaction  to  the 
stain  is  the  same  as  in  the  cross  section. 

Study  and  sketch  as  seen  under  high  power. 
DRAWINGS  FOR  LESSON  XIII. 


- 


DRAWINGS  FOR  LESSON  XJHr 


'f 


C 


—  53  — 


LESSON  XIV. 
NERVE  CELLS. 

(a)  Nerve  Cells  from  Spinal  Cord. 

The  gray  matter  was  dissected  from  the  cervical  cord  of  an  ox, 
macerated  for  several  days  in  a  very  weak  solution  of  chromic  acid 
(1-15,000),  and  was  then  stained  in  lithium  carmine.  Tease  very 
carefully  in  gum  glycerine,  controlling  the  results  under  the  low 
power.  Aim  to  isolate  the  cells  from  the  surrounding  tissue.  The 
worth  of  this  preparation  will  depend  very  largely  on  the  care  with 
which  it  is  teased. 

Examine  under  high  power,  and  observe  the  large 
branching  nerve  cells;  try  to  make  out  the  axis  cylinder 
process.  Sketch  several  cells,  under  this  power. 

(b)  Nerve  Cells  from  the  Spinal  Cord  Stained  in 

Methylene  Blue. 

The  gray  matter  was  dissected  from  a  spinal  cord,  and  crushed 
between  two  cover  glasses,  these  are  then  drawn  apart,  and  set 
aside  to  dry.  Stain  for  30  minutes  in  a  1%  methylene  blue  solution, 
wash  in  water,  and  dry  between  filter  paper.  When  thoroughly  dry 
mount  the  preparation  in  balsam. 

Study  the  preparation  under  high  power.  A  few 
nerve  cells  will  be  found.  Observe  the  relatively  large 
nucleus  with  its  prominent  nucleolus;  also  the  spongio- 
blastic  network  in  the  cell  body.  Sketch  as  seen  under 
high  power. 

(c)  Spinal  Cord  Stained  after  Golgi's  Method. 

The  spinal  cord  of  an  embryo  or  young  mammal  was  divided 
into  small  pieces,  placed  for  2  or  3  days  in  a  solution  composed  of  1 
part  of  a  \%  solution  of  osmic  acid  and  4  parts  of  a  3%  solution  of 
potassium  bichromate,  then  in  a  %%  solution  of  silver  nitrate  for 
about  an  hour,  finally  in  a  %%  solution  of  the  same  salt  in  which 
they  remained  for  2  to  4  days,  then  surrounded  with  paraffin,  and 
sectioned.  The  sections  were  cut  into  95%  alcohol,  transferred  to 


\ 


—  54  — 

creosote  for  15  minutes,  and  then  into  turpentine.  Transfer  a  sec- 
tion to  the  slide,  remove  as  much  of  the  turpentine  as  possible, 
cover  with  a  large  drop  of  balsam,,  and  heat  the  slide  over  the  flame  for 
several  minutes.  Cover  with  a  warm  cover  glass,  and  set  aside  to  cool. 

Study  under  high  power.  Only  a  few  of  the  nerve 
cells  are  stained  (a  brownish  black),  but  these  are  usually 
brought  out  very  clearly.  The  protoplasmic  processes  are 
seen  branching  and  rebranching,  and  the  axis  cylinder  pro- 
cess may  now  and  then  be  traced  through  several  fields  of 
the  microscope.  This  process  gives  off  only  a  few  small 
branches.  A  few  neuroglia  cells  may  also  be  stained; 
they  have  a  small  body  from  which  many  fine  processes 
radiate.  Sketch  nerve  and  neuroglia  cells  as  seen  under 
high  power. 

(d)  Brain  Cortex  Stained  after  Golgi's  Method. 

These  sections  were  stained  according  to  the  above  mentioned 
method.  Mount  in  the  same  way  as  the  spinal  cord  section. 

Study  under  high  power.  The  cortical  cells  have  a 
triangle-shaped  body ;  an  axis  cylinder  process  is  given 
off  from  the  base,  which  passes  down  into  the  white  sub- 
stance; the  protoplasmic  processes  are  large,  especially 
the  one  given  off  from  the  apex.  Sketch  a  number  of  the 
cells  as  seen  under  high  power. 

(e)  Cerebellum  Stained  after  Golgi's  Method. 

The  section  was  stained  as  above ;  mount  in  hard  balsam  as 
directed. 

Study  first  under  low  power,  and  search  for  Purkinje's 
cells.  These  are  often  very  beautifully  stained.  They 
have  an  oval  or  pear-shaped  body,  from  the  lower  end  of 
which  an  axis  cylinder  process  is  given  off,  and  from  th3 
other  end  a  large  protoplasmic  process  which  usually 
divides  into  two  or  three  branches  and  these  divide  and 
redivide  until  a  complicated  system  of  branches  is  formed. 
A  few  of  the  nerve  cells  in  the  granular  layer  and  some  of 
the  basket  cells  may  also  be  stained.  Sketch  the  cells 
under  high  power. 


_         £  r    

(f  J  Posterior  Root  Ganglion  Stained  in  Osmic  Acid. 

The  posterior  root  ganglion  of  a  small  mammal  was  hardened 
in  llenirum's  solution,  embedded  in  paraffin,  and  sectioned  longi- 
tudinally. Fix  the  section  to  the  slide,  and  mount  in  balsam. 

Study  under  low  power.  Observe  that  the  fibrous  cap- 
sule around  the  ganglion  is  continuous  with  the  epineurium 
of  the  in-coming  and  out-going  nerve  trunk.  The  nerve 
fibres  are  stained  black.  The  nerve  trunk  breaks  up  into 
several  smaller  bundles  after  it  enters  the  ganglion.  Be- 
tween these  bundles  may  be  seen  groups  of  ganglion  cells, 
round  or  oval  in  shape,  with  prominent  nuclei  and  nucleoli. 
They  are  stained  a  faint  brown  color.  The  cells  are  sur- 
rounded by  a  nucleated  capsule.  Sketch  the  ganglion 
under  low  power. 

(g)  Postarior  Root  GFinglion   Stained  in  Hsema- 
toxylin. 

The  posterior  root  iranglion  of  a  dog  was  hardened  in  mercuric 
chloride,  stained  in  Delafield's  hsematoxylin,  embedded  in  paraffin, 
and  sectioned  longitudinally.  Fix  to  the  slide,  and  mount  in  balsam. 

Study  first  under  low  power,  and  observe  the  structure 
as  described  for  (f)  of  this  lesson.  Sketch  a  number  of 
the  cells  with  the  intervening  nerve  fibres  as  seen  under 
high  power. 

DRAWINGS  FOR  LESSON  XIV. 


DRAWINGS  FOR  LESSON  XIV, 


—  57  — 


LESSON  XV. 

SPINAL,  CORD,  CEREBELLUM,  AND 
CEREBRUM. 

(a)  Spinal    Cord   with    Anterior    and    Posterior 

Roots. 

A  segment  of  the  cervical  cord  of  a  cat  with  the  anterior  and 
posterior  roots  of  the  two  .sides  (to  a  point  a  short  distance  beyond 
their  junction)  was  removed  and  hardened  in  mercuric  chloride. 
To  insure  the  hardening  of  the  roots  in  a  plane  so  that,  the  cord  and 
roots  on  both  sides  might  be  cut  in  one  section,  the  following  de- 
vice was  made  use  of:  An  opening  large  enough  to  admit  the  cord 
was  made  in  a  card;  one  end  of  the  cord  segment  was  pushed 
through  this  opening,  and  the  roots  were  spread  out  on  the  card  to 
which  they  adhered.  The  preparation  was  then  placed  in  mercuric 
chloride.  After  hardening  it  was  stained  en  masse  in  borax  carmine 
for  several  days,  embedded  in  paraffin,  and  sectioned.  Fix  to  the 
slide,  and  mount  in  balsam. 

Study  under  low  power.  Observe  the  anterior  and  pos- 
terior roots  in  connection  with  the  anterior  and  posterior 
horns  of  the  gray  matter.  .  Sketch  under  low  power. 

(b)  Section  of  Spinal  Cord  Stained  in  Weigert's 

Hsematoxy  lin . 

A  human  cord  was  hardened  in  Mailer's  fluid,  embedded  in 
celloidin,  stained  in  Weigert's  hsematoxylin,  dehydrated,  and 
cleared  in  carbol-xylol.  Place  section  on  slide, 'wipe  away  excess  of 
clearing  fluid,  and  mount  in  balsam. 

Study  under  low  power.  The  cord  is  nearly  divided 
into  two  symmetrical  halves  by  two  fissures,  the  anterior 
and  posterior  median  fissures,  the  latter  is  really  only  a 
connective  tissue  septum.  In  each  half  of  the  cord  the 
gray  matter,  which  in  this  section,  has  a  yellowish-brown 
color,  appears  in  the  form  of  a  crescent.  The  convex 
borders  of  the  two  crescents  are  opposed  to  each  other. 
The  anterior  horns  of  gray  matter  are  broader  and  shorter, 


-58- 

and  do  not  come  so  near  to  the  surface  as  the  posterior. 
The  two  crescents  are  united  by  an  anterior  white,  and  a 
posterior  gray,  commissure,  between  which  is  found  the 
central  canal.  In  the  gray  matter  a  very  fine  network  of 
fibres  is  seen.  This  is  composed  of  medullated  fibres, 
branches  of  nerve  cells,  and  neuroglia  tissue.  The  nerve 
cells  are  usually  found  in  small  groups,  those  in  the  ante- 
rior horn  are  larger  and  more  numerous.  The  white  mat- 
ter, stained  a  bluish  black,  surrounds  the  gray.  It  is  corn- 
posed  of  rnedullated  nerve  fibres  seen  in  cross  section. 
Between  these  fibres  a  small  amount  of  neuroglia  tissue  is 
observed.  Sketch  the  cord  in  outline  as  seen  under  low 
power,  and  sketch  portions  of  the  gray  matter  of  the 
anterior  horn  and  white  matter  as  seen  under  high  power. 

(c)  Cerebellum. 

Small  pieces  of  the  cerebellum  were  hardened  in  mercuric 
chloride,  stained  in  Delafield's  heematoxylin,  embedded  in  pnraflin, 
and  sectioned  at  right  angles  to  the  small  folds.  Fix  to  the  slide 
and  mount  in  balsam. 

Under  high  power  the  gray  matter  shows  the  follow- 
ing layers  in  cross-section  : — 

(1)  An  outer   molecular  layer,  composed    largely    of 
neuroglial  tissue,  and  containing  a  few  small  ganglion  cells. 

(2)  Between  the  above  stratum  and  the  third  a  single 
layer  of  Purkinje's  cells   is  found.     From  the  base  of  these 
cells  an  axis  cylinder  process  is  given  off;  from  the  oppo- 
site pole  one  or  two  protoplasmic  processes,  these  extending 
into  the  molecular  layer  and  there  dividing  and  redividing, 
until  the  process  has  the  appearance  of  a  deer's  antlers. 

(3)  The    granular   layer,  composed  largely  of  round 
and  spindle-shaped    cells    possessing   comparatively  large 
nuclei,  so  that  in  the  section  very  little  but  the  nuclei  will 
be  seen.    The  axis  cylinder  processes  of  Purkinje's  cells  pass 
through  this  layer,  become  medullated,  and  are  lost  in  the 
white  substance  making  up  the  central  portion  of  the  fold. 
Sketch  the  cortex  under  high  power. 

(d)  Cerebral  Cortex. 

Small  pieces  of  cerebral  cortex  were  hardened  in  mercuric 
chloride,  stained  in  Delafield's  hsematoxylin,  embedded  in  paraffin, 


—  59  — 

and  sectioned  at  right  angles  to  the  surface.  Fix  and  mount  in 
balsam. 

Under  high  power  the  following  structure  will  be 
seen : — 

(1)  The  outer  or  molecular  layer,  composed  largely 
of  neuroglial  tissue.  In  it  are  a  few  small  nerve  cells  and 
a  thin  stratum  of  fine  medullated  fibres,  running  parallel 
to  the  surface  just  under  the  pia  mater. 

(2  and  3)  Layer  of  small  and  large  pyramidal  cells. 
In  the  former  layer  the  cells  are  small  and  close  together; 
in  the  latter  they  are  larger  and  farther  apart.  There  is, 
however,  no  distinct  boundary  line  between  the  two  layers. 

(4)  A  layer  of  small  cells,  some  of  which  are  pyram- 
idal, others  spindle-shaped,  still  others  multipolar. 

Sketch  a  portion  of  the  cortex  under  high  power. 

(e)  Cerebral  Cortex  Stained  with  Weigert's  Haem- 
atoxylin. 

Small  pieces  of  the  human  cortex  were  hardened  in  Muller'  s 
fluid,  embedded  in  celloidin,  sectioned  at  right  angles  to  the  surface, 
stained  in  Weitrert's  hsematoxylin,  dehydrated,  and  cleared  in 
carbol-xylol.  Wipe  away  excess  of  clearing  fluid,  and  mount  in 
balsam.  The  nerve  fibres  are  stained  a  bluish-black,  all  other 
elements  (except  the  red  blood  cells)  a  yellowish  brown. 

Study  under  low  power.  "The  nerve  fibres  entering 
the  gray  cortex  are  arranged  in  bundles,  from  which  arise 
networks  variously  arranged  and  situated.  The  radiating 
bundles  proceed  as  such  through  about  half  the  entire 
thickness  of  the  cortex;  beyond  this  level  they  rapidly 
separate  into  the  component  ..fibres  which  take  their  way 
between  the  ganglion  cells."  (Piersol.)  Sketch  a  portion 
of  the  cortex  as  seen  under  low  power. 


. 


DRAWINGS  FOR  LESSON  XV. 


DRAWINGS  FOR  LESSON  XV. 


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DRAWINGS  FOR  LESSON  XV. 


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. 


—  63  — 


LESSON  XVI. 
BLOOD  VESSELS. 

(a)  Capillaries. 

The  thoracic  aorta  of  a  rat  or  guinea  pig  was  injected  vvitli  a 
\%  solution  of  silver  nitrate.  After  20  to  30  minutes  the  mesentery 
was  removed,  pinned  out  on  flat  corks,  placed  in  95%  alcohol,  in 
which  the  tissue  was  exposed  to  direct  sunlight.  In  a  few  minutes 
the  tissue  turns  a  brownish  color.  It  was  then  dehydrated,  cleared 
in  oil  of  bergamot,-and  cut  into  small  pieces.  Mount  in  baisntn.  •-.'*•,  c^^ 

Study  under  high  power.  Small  capillaries  and  arter- 
ioles,  the  endothelial  cells  of  which  are  outlined  by 
brownish-black  lines,  will  be  seen.  Sketch  a  number  of 
the  capillaries  under  high  power. 

(b)  Small  Arterioles. 

The  mesentery  of  a  cat  was  pinned  out  on  a  cork,  hardened  in 
alcohol,  stained  in  Delafield's  hsematoxylin,  dehydrated,  and  cleared 
in  oil  of  bergainot.  Mount  in  balsam. 

Study  under  high  power.  Look  for  arterioles  consist- 
ing of  an  endothelial  coat,  and  a  media  composed  of  a 
single  or  double  layer  of  non-striped  muscle  cells,  about 
which  there  is  a  thin  layer  of  fibrous  tissue.  Search  for 
capillaries,  in  which  the  nuclei  of  the  endothelial  cells  are 
stained.  Sketch  an  arteriole  and  capillary  as  seen  under 
high  power. 

(c)  Artery  and  Vein. 

The  posterior  tibial  artery  and  vein  (human)  were  hardened 
in  mercuric  chloride  or  alcohol,  stained  in  borax  carmine  or  Dela- 
field's hajMiatoxylin,  embedded  in  paraffin,  and  cross-sectioned. 
Fix  to  the  slide  and  mount  in  balsam. 

Study  first  under  low  power.  In  the  wall  of  the  artery 
three  coats  are  seen, — 


—  64  — 

(J)  An  inner  coat,  the  tunica  intima,  which  consists 
of  a  single  layer  of  endothelial  cells  lining  the  lumen  of 
the  vessel,  a  thin  stratum  of  sub-endothelial  connective 
tissue,  and  a  stratum  of  elastic  tissue,  the  elastic  intima  or 
ftnestrated  membrane  of  Henle. 

(2)  The  middle  coat,  tunica  media,  composed  largely 
of  circularly  disposed  bundles  of  non-striped  muscle  tissue  ; 
between  these  thin  films  of  elastic  tissue,   seen  in  cross 
section  as  wavy  lines,  are  found. 

(3)  The    outer    coat,    tunica    adventitia,    consists    of 
bundles  of  white  fibrous  tissue  felted  into  a  dense  network. 
Between  these  bundles  a  few  elastic  fibres  are  seen.     This 
coat  is  continuous  with  the  surrounding  connective  tissue. 

The  wall  of  the  vein  is  not  so  thick.  Three  coats  are 
seen,  resembling  in  structure  the  ones  seen  in  the  artery. 
In  the  intima  of  the  vein  the  elastic  tissue  is  not  so  prom- 
inent ;  in  the  tunica  media  there  is  relatively  less  muscle ; 
the  bundles  of  muscle  are  separated  by  white  fibrous  tissue. 
The  adventitia  is  thicker  .than  the  corresponding  coat  of 
the  artery.  This  section  also  shows  in  section  a  number 
of  small  arteries  and  veins  in  the  fibrous  tissue  surround- 
ing the  larger  vessels. 

Sketch  a  segment  of  the  arterial  wall,  also  of  the 
venous  wall/ and  sketch  a  small  artery,  all  under  low  power. 

(d)  Aorta. 

Small  pieces  of  the  aorta  were  hardened  in  alcohol,  stained  in 
borax  carmine,  embedded  in  paraffin,  and  cross-sectioned.  Fix  and 
mount  in  balsam. 

The  media  is  relatively  thick,  and  is  composed  of 
alternating  layers  of  non-striped  muscle  and  yellow  elastic 
tissue.  The  majority  of  the  non-striped  muscle  cells  are 
arranged  transversely,  some  few  longitudinally.  The  adven- 
titia is  a  relatively  thin  coat,  composed  largely  of  white 
fibrous  tissue,  together  with  a  few  elastic  and  non-striped 
muscle  fibres.  The  intima  is  composed  of  endothelial  cells, 
elastic,  and  white  fibrous  tissue.  Sketch  a  portion  of  the 
wall  under  high  power. 


DRAWINGS  FOR  LESSON  XVI. 


DRAWINGS  FOR  LESSON  XVI. 


—  67- 


LESSON  XVII. 

SIMPLE    ADENOID   TISSUE.  THYMUS, 
LYMPH  GLAND,  AND  SPLEEN. 

(a)  Simple  or  Diffuse  Adenoid  Tissue. 

A  portion  of  the  vermiform  appendix  of  a  rabbit  was  hardened 
in  absolute  alcohol,  stained  in  borax  carmine,  embedded  in  paraffin, 
and  cross-sectioned.  Fix  to  the  slide,  and  mount  in  balsam. 

Examine  under  high  power.  The  mucosa  and  sub- 
mucosa  form  a  continuous  zone  of  adenoid  tissue.  The 
mass  of  round  cells  are  so  closely  packed  that  they  cover 
up  the  adenoid  reticulum  in  which  they  are  found.  Sketch 
as  seen  under  high  power. 

(b)  Tonsil. 

The  tonsil  of  a  dog  was  hardened  in  mercuric  chloride,  stained 
in  Delafield's  hsematoxyliu,  embedded  in  paraffin,  and  sectioned. 
Fix  and  mount  in  balsam. 

Study  first  under  low  power.  Under  the  stratified 
pavement  epithelium  are  found  a  number  of  round  or  oval 
nodules  of  adenoid  tissue  embedded  in  diffuse  adenoid  tis- 
sue. They  are  more  or  less  completely  separated  from  the 
surrounding  tissue  by  a  fibrous  tissue  envelope.  Under 
high  power  it  will  be  noticed  that  in  many  places  cells  from 
the  adenoid  tissue  have  wandered  into  the  epithelium,  often 
to  such  an  extent  that  it  is  difficult  to  recognize  the  epithe- 
lial cells.  One  or  more  small  mucous  glands  are  often  seen 
in  sections  of  the  tonsil.  Sketch  under  low  power. 

(c)  Thymus  Gland. 

The  thyrnus  gland  of  a  new-born  mammal  was  hardened  in 
mercuric  chloride,  stained  in  Delafield's  hiematoxylin,  embedded  in 
paraffin,  and  sectioned.  Fix  and  mount  in  balsam. 

Study  under  low  power.     The  gland  is  divided  into 


—  68  — 

lobules  which  are  surrounded  by  fibrous  tissue,  from  which 
septa  pass  into  the  lobules  and  divide  them  into  secondary 
lobules.  The  secondary  lobules  are  composed  of  adenoid 
tissue.  This  is  denser  and  stains  more  deeply  in  the  per- 
iphery of  the  lobules  forming  the  cortical  zone;  the  less 
dense  center  forms  the  medullary  portion.  In  the  medul- 
lary portion  are  found  the  concentric  corpuscles  of  Hassall. 
They  are  the  remains  of  the  entodermal  epithelial  tissue  of 
which  the  gland  was  composed  in  its  early  development. 

(d)  Compound  Lymph  Gland. 

A  tyinph  gland  was  hardened  in  mercuric  chloride,  stained  in 
Delafield's  hsematoxylin,  embedded  in  paraffin,  and  sectioned.  S^c- 
tions  were  fixed  to  cover  glasses,  counter-stained  in  eosin,  dehy- 
drated, and  cleared  in  oil  of  ber^amot  and  xylol.  Mount  on  a  drop 
of  balsam. 

Examine  first  under  low  power.  Observe  the  capsule, 
from  which  trabeculse  pass  into  the  gland,  dividing  its  outer 
or  cortical  portion  into  comparatively  large  compartments, 
in  which  the  cortical  nodules  of  adenoid  tissue  are  found. 
On  entering  the  medulla  the  trabecula?  divide  and  anasto- 
mose ;  the  network  so  formed  has  small  meshes  in  which 
are  found  the  medullary  cylinders  of  adenoid  tissue.  The 
adenoid  tissue  is  composed  of  a  reticular  framework  and 
cells. 

"  The  cortical  follicles  and  the  medullary  cylinders  do 
not  completely  fill  out  the  compartments  made  for  them 
b\r  the  capsule  and  trabecula3  respectively,  but  a  narrow  per- 
ipheral zone  of  each  compartment  is  left  free,  this  is  a 
lymph  sinus."  (Klein.)  The  lymph  sinuses  form  an  anas- 
tomosing system.  Sketch  a  portion  of  the  gland  under  low 
power. 

(e)  Reticulum  of  Adenoid  Tissue. 

A  lymph  gland  was  hardened  in  alcohol,  and  cut  on  the  freez- 
ing microtome.  The  sections  were  stained  in  Boehmer's  hsematoxy- 
lin.  They  were  then  transferred  to  a  test-tube  half  full  of  distilled 
water,  in  which  they  were  carefully  shaken  for  10  or  15  minutes.  In 
this  way  many  of  the  lymph  cells  are  shaken  out  of  the  reticulum. 
Mount  in  gum  gtycerine. 

Examine  under  high  power,  using  one  of  the  smaller 
openings  in  the  diaphragm.  A  reticulum  of  very  fine 


-69- 

fibrils  will  be  seen.     Fixed  connective  tissue  cells  are  often 
found  on  the  network.     Sketch  under  this  power. 

(f )  Lymph  Cells. 

Cover  glasses  were  drawn  over  the  freshly  cut  surface  of  a  lymph 
gland,  and  fixed  in  a  solution  of  3  parts  ether  and  1  part  absolute 
alcohol  for  %  hour.  Stain  in  haematoxylm  for  15  minutes,  wash  in 
water,  counter-stain  in  eosin  for  5  minutes,  again  wash  in  water, 
and  dry  between  filter  paper.  Mount  in  balsam. 

Study  under  high  power.  The  greater  proportion  of 
the  cells  seen  have  the  shape  and  structure  of  the  lympho- 
cytes found  in  the  blood.  A  few  mononuclear  and  polynu- 
clear  cells  will  also  be  found.  Sketch  under  high  power. 

(g)  Spleen. 

Small  pieces  from  the  spleen  of  a  dog  were  hardened  in  mer- 
curic chlorid^,  stained  in  borax  carmine,  embedded  in  paraffin,  and 
sectioned.  Fix  to  the  slide,  and  mount  in  balsam. 

Observe  the  capsule  composed  of  connective  tissue  and 
non-striped  muscle  cells.  From  the  capsule  trabeculse 
pass  into  the  gland,  branching  and  forming  an  anastomos- 
ing network ;  their  structure  is  similar  to  that  of  the  cap- 
sule. Within  the  capsule  two  kinds  of  tissue  are  found, 
the  Malpighiau  corpuscles  and  the  splenic  pulp.  The 
former  are  composed  of  adenoid  tissue,  and  are  usually 
found  surrounding  the  vessels.  In  the  pulp  a  framework 
of  fine  fibrils  and  cells  is  found,  in  the  meshes  of  which 
red  and  white  blood  cells  are  seen.  Sketch  a  portion  under 
high  power. 

(h)  Cover  Glass  Preparation  of  Splenic  Pulp. 

Cover  glasses  were  drawn  over  the  freshly  cut  surface  of  a 
spleen,  and  fixed  in  ether-alcohol  solution  for  1.  hour.  Stain  in 
hsematoxylin  and  eosin  as  (f )  of  this  lesson,  and  mount  on  balsam. 

Study  under  high  power.  Many  red  blood  cells  stained 
with  the  eosin,  and  the  various  forms  of  white  blood  cells, 
will  be  seen.  Of  the  white  blood  cells  the  mononuclear 
are  found  in  much  larger  proportion  than  in  circulating 
blood.  Sketch  the  cells  as  seen  under  high  power. 


DRAWINGS  FOR  LESSON  XVII. 


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DRAAVINGS  FOR  LESSON  XVII. 


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DRAWINGS  FOR  LESSON  XVIII 


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—  73  — 


LESSON  XVIII. 
SKIN  AND  APPENDAGES. 

(a)  Macerated  Epidermis. 

A  small  piece  of  skin  was  macerated  for  several  days  in  %% 
solution  of  acetic  acid.  The  epidermis  was  then  carefully  lifted 
from  the  dermis,  rinsed  in  water,  hardened  in  alcohol,  stained  in 
borax  carmine,  and  cleared  in  oil  of  bergamot.  Mount  in  balsam, 
taking  care  to  mount  it  in  such  a  way  that  the  under  surface  of  the 
epidermis  is  uppermost. 

Study  under  low  power.  Observe  the  depressions  in 
the  under  surface  of  the  epidermis  into  which  the  papillae 
of  the  true  skin  fit.  Note  their  number  and  arrangement. 
Sketch  under  low  power. 

(b)  Preparation  of  the  Dermis. 

The  skin  was  macerated  as  above,  and  the  epidermis  removed. 
The  dermis  was  hardened  in  alcohol,  stained  in  borax  carmine,  em- 
bedded in  paraffin,  and  sectioned. 

Study  under  low  power.  This  section  is  given  to  show 
the  papillae  of  the  true  skin.  In  many  of  these  a  capil- 
lary network  can  be  made  out.  Sketch  a  number  of  the 
papillae  under  low  power. 

(cj  Cross-Section  of  Human  Skin. 

A  small  piece  of  skin  removed  from  the  plantar  surface  of  the 
foot  or  palmar  surface  of  the  hand  was  hardened  in  10%  nitric  acid 
for  24  hours,  then  in  Muller's  fluid  for  two  weeks,  stained  in  Dela- 
field's  haematoxylin,  embedded  in  paraffin,  sectioned,  fixed  to  cover 
glasses,  counter-stained  in  \%  acid  fuchsin,  dehydrated,  and  cleared 
in  oil  of  bergamot  and  xylol.  Mount  in  balsam. 

This  section  is  to  be  studied  first  under  low  power,  and 
the  general  arrangement  of  the  tissues  observed.  In  the 
epidermis  the  following  layers  are  found,  named  in  order 
from  within,  outward  : — 


—  74  — 

(1)  Stratum   Malpighii   (rete  mucosa),    composed  of 
stratified  pavement  epithelium. 

(2)  Stratum    granulosum,    a    narrow    layer    of     cells 
which  contain  kerato-hyalin  granules  (Waldeyer)  in  their 
protoplasm. 

(3)  Stratum  lucidum,  composed   of    horny   cells  the 
outlines  of  which  are  very  indistinct. 

(4)  Stratum  corneum,  the  thick  outer  layer. 
Observe  also  the  twisted  portion  of    the  sudoriferous 

glands  passing  through  the  epidermis. 

In  the  cutis  vera  an  outer  denser  portion,  the  papillary 
layer,  is  observed.  This  is  composed  of  closely  interwoven 
bundles  of  white  fibrous  tissue,  between  which  a  small 
amount  of  elastic  tissue  is  found.  This  layer  is  beset  with 
papillae.  In  the  deeper  portion  of  the  corium,  the  reticular 
layer,  the  bundles  of  fibrous  tissue  are  loosely  woven.  In 
the  large  open  meshes  groups  of  fat  cells  and  the  secreting 
portion  of  the  sweat  glands  are  found.  Some  of  the  prep- 
arations may  show  a  Vater-Pacinian  corpuscle  in  section. 

Sketch  the  several  layers  of  the  epidermis  and  the 
dermis  as  seen  under  low  power. 

(d)  Cross-Section  of  the  Scalp. 

A  portion  of  the  scalp  was  hardened  in  Miiller's  fluid,  embed- 
ded in  celloidin,  sectioned,  and  stained  in  hseinatoxylin  and  eosin. 
The  sections  are  in  oil,  and  are  to  be  mounted  in  balsam. 

In  this  section  special  attention  is  to  be  given  to  the 
study  of  the  hair  and  its  follicle.  An  attempt  was  made 
to  cut  them  longitudinally.  Examine  first  under  low 
power,  and  observe  the  several  layers  of  the  follicle,  the 
sebaceous  gland  seen  in  connection  with  it,  and  the 
arrectores  pili.  Sketch  under  this  power. 

(e)  Tangential  Section  of  the  Scalp. 

This  tissue  was  prepared  as  the  cross-section.  Mount  in  balsam. 

In  this  section  the  hair  follicles  are  seen  in  cross-sec- 
tion. Study  under  high  power. 

Each  follicle  shows  the  following  layers,  named  in 
order  from  without,  in  : — 


In  the  dermal  coat, 

(1)  A  longitudinal  fibrous  layer. 

(2)  A  circular  fibrous  layer. 

(3)  Glassy  layer. 

In  the  epidermal  coat, 

(1)  Outer  root  sheath  (rete  mucosa). 

(2)  Henle's  layer. 

(3)  Huxley's  layer. 

(4)  Cuticle  of  the  shaft. 

Sketch  a  follicle  as  seen  under  high  power. 

DRAWINGS  FOR  LESSON  XVIII. 


« 

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DRAWINGS  FOR  LESSON  XVIII. 


—  77  — 


LESSON  XIX. 
THE  DIGESTIVE  TRACT. 

(a)  Cross-Section  of  Human  Lip. 

Pieces  of  the  lip  of  a  child  were  hardened  in  mercuric  chloride, 
stained  in  Delafield's  hsematoxylin,  embedded  in  paraffin,  and 
cross-sectioned.  Fix  to  the  slide  and  mount. 

Study  under  low  power.  Observe  that  the  outer  sur- 
face of  the  lip  is  covered  with  a  thin  epidermis  resting  on 
a  dermis  in  which  many  hair  follicles,  sebaceous  and  sweat 
glands  are  found.  The  inner  surface  of  the  lip  is  covered 
with  a  mucous  membrane  consisting  of  stratified  pavement 
epithelium  and  a  fibrous  mucosa.  In  the  submucous 
tissue,  and  in  the  deeper  parts  of  the  mucosa,  are  often 
found  quite  large  mucous  glands  which  empty  on  the  inner 
side  of  the  lip.  Between  the  epidermal  and  mucous  sur- 
faces striped  muscle,  fibrous,  and  adipose  tissue  is  found. 
Often  one  or  more  quite  large  blood  vessels  and  nerve 
trunks  are  seen.  Sketch  under  low  power. 

(b)  Section    of  the    Tongue    Showing     Filiform 

Papillae. 

The  tip  of  the  tongue  of  a  cat  was  hardened  in  mercuric 
chloride,  stained  in  Delafield's  hsematoxylin,  embedded  in  paraffin, 
and  longitudinal  sections  cut.  Fix  to  the  slide,  and  mount  in 
balsam. 

Study  under  low  power.  The  papilla?  are  found  on 
the  dorsal  surface.  The  mucosa  is  thrown  into  conical 
projections,  the  primary  papillae,  on  which  several  second- 
ary papillae  are  often  seen.  The  papillae  are  covered  with 
stratified  pavement  epithelium,  which  on  the  outer  surface 
has  a  smooth  free  border.  Sketch  several  papilla?  under 
high  power. 


-78- 

(c)  Section  of  Tongue  showing  Fungiform  Papil- 

Ue. 

Portions  from  the  posterior  part  of  a  dog's  tongue,  known  to, 
contain  fuugiform  papillae,  were  hardened  in  mercuric  chloride, 
stained  in  Pelnfield!s  hsematoxylin,  embedded  in  paraffin,  and  sec- 
tioned at  right  angles  to  the  long  axis  of  the  tongue.  The  sections 
were  fixed  to  cover  glasses  and  counter  stained  in  eosin,  dehydrated, 
and  cleared  in  oil  of  bergamot  and  xylol.  Mount  on  balsam. 

Study  first  under  low  power.  Search  for  the  broad 
conical  projections  of  the  mucosa,  composed  of  a  rather 
dense  fibrous  tissue  on  which  many  secondary  papillae  are 
seen.  They  are  covered  with  stratified  pavement  epithe- 
lium. Many  fillit'orm  papillae  will  also  be  seen  in  this 
section.  Observe  the  arrangement  of  the  muscle  fibres. 
The  preparation  will  also  show  arteries,  veins,  and  nerves 
in  section.  Sketch  one  fungiform  papilla  as  seen  under 
high  power. 

(d)  Taste  Buds. 

The  papilla  foliata  of  a  rabbit  was  hardened  in  mercuric 
chloride,  stained  in  Delafield's  hsematoxylin,  embedded  in  paraffin, 
and  sectioned  at  right  angles  to  the  transverse  ridgps  seen  on  the 
papilla.  Mount  in  balsam. 

The  taste  buds  are  embedded  in  the  epithelium  lining 
the  furrows  between  the  ridges.  In  the  muscular  and  con- 
nective tissue  below  the  papilla  foliata  small  serous  glands 
will  be  seen  in  section.  Sketch  two  ridges  with  the  inter- 
vening furrow  and  taste  buds  as  seen  under  high  power. 

(e)  Mucous  and  Serous  Salivary  Glands. 

Small  pieces  taken  from  the  submaxillary  and  parotid  glands 
of  a  dog  were  hardened  in  murcuric  chloride,  pieces  of  each  gland 
embedded  side  by  side  in  paraffin  (so  that  one  section  will  pass 
through  both  glands),  and  sectioned.  The  sections  were  then  fixed 
to  cover  glasses,  stained  in  Haidenhain's  iron-lack-hgematoxylin, 
dehydrated,  and  cleared  in  oil  of  bergamot  and  xylol.  Thus  on 
each  cover  glass  are  sections  of  both  submaxillary  and  parotid 
glands.  Mount  in  balsam. 

Study  first  under  low  power.  The  less  deeply  stained 
section  is  the  mucous  (submaxillary)  gland.  Both  glands 
have  the  same  general  structure.  The  acini  are  grouped 
into  lobules  which  are  held  together  by  a  loose  fibrous  tis- 


—  79  — 

sue.  The  acini  of  the  mucous  glands  are  larger  than  those 
of  the  serous  glands.  Study  the  structure  of  the  acini 
under  high  power. 

In  the  mucous  glands  the  acini  are  surrounded  by  a 
membrana  propria  which  is  nearly  filled  with  clear  mucous 
cells  stained  a  steel-gra}^.  The  nuclei  are  in  the  peripheral 
part  of  the  cells.  Many  of  the  acini  will  show  a  crescent 
of  more  deeply  stained  cells,  the  demilunes  of  Heidenhain, 
lying  between  the  mucous  cells  and  the  membrana  propria. 
The  ducts  are  regularly  round  or  oval  with  relatively  large 
lumina.  They  are  lined  by  a  single  layer  of  columnar 
cells  with  striated  protoplasm. 

The  acini  of  the  serous  glands  also  have  a  limiting 
membrana  propria.  They  are  lined  by  cuboidal  or 
polyhedral  cells  with  the  nuclei  near  the  center  of  the 
cells.  They  often  have  a  granular  protoplasm.  The 
structure  of  the  ducts  is  the  same  as  in  the  mucous  glands. 

Sketch  a  duct  and  a  number  of  acini  surrounding  it, 
from  both  mucous  and  serous  glands  as  seen  under  high 
power. 

(f)  Mucous  and  Serous  Salivary  Glands  Stained 

in  Delafield's  Haematoxylin. 

Pieces  of  the  submaxillary  and  parotid  glands  of  a  dog  were 
hardened  in  mercuric,  chloride,  stained  in  Delafield's  hsematoxylin, 
and  a  piece  of  each  gland  embedded  side  by  side  in  a  block  of 
paraffin,  and  sectioned.  Fix  to  the  slide  and  mount  in  balsam. 

The  structure  seen  in  (e)  will  again  be  observed. 

(g)  Mucous  and  Serous  Salivary  Glands  Stained 

in  Borax  Carmine. 

Pieces  from  the  submaxillary  and  parotid  glands  of  a  dog  were 
hardened  in  absolute  alcohol,  stained  in  borax  carmine,  pieces  of 
each  gland  embedded  as  above,  and  sectioned.  Fix  and  mount. 

The  last  two  preparations  are  given  to  the  student  to 
show  the  structure  of  mucous  and  serous  glands  as  shown 
by  ruematoxylin  and  carmine  stains  to  facilitate  their 
recognition  in  sections  to  be  given  later  on  in  the  work. 


DRAWINGS  FOR  LESSON  XIX. 


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DRAWINGS  FOR  LESSON  XIX. 


-83  — 


LESSON^  XX. 
DIGESTIVE  TRACT  (Continued). 

(a)  Longitudinal  Section  of  Hard  Tooth. 

By  means  of  a  fine  saw  a  longitudinal  section  is  cut  from  a 
tooth  (incisors  or  canines  best).  Grind  this  on  an  emery  wheel  as 
thin  as  possible,  and  then  between  two  hones  until  it  becomes  quite 
transparent.  Care  must  be  taken  to  grind  it  evenly  ;  keep  the 
hones  well  moistened  with  water.  Wash  thoroughly,  first  in  water, 
then  in  alcohol ;  then  dry  the  section  between  filter  paper.  Mount 
in  hard  balsam. 

Study  the  preparation  first  .under  low  power,  observing 
the  shape  and  size  of  the  pulp  cavity,  and  the  relative  pro- 
portion of  the  dentine,  cement,  and  enamel.  The  structure 
of  these  parts  is  then  to  be  studied  under  high  power.  In 
the  enamel  note  the  enamel  prisms;  in  the  dentine  the 
dentinal  tubules  radiating  from  the  pulp  cavity,  and  in  its 
peripheral  portion,  the  interglobular  spaces.  The  cement 
shows  the  structure  of  bone;  very  seldom,  however,  are 
Haversian  canals  found.  Sketch  under  low  power. 

(b)  Cross  Section  of  Decalcified  Tooth. 

The  canine  of  a  young  cat  was  extracted,  the  pulp  cavity 
opened  from  above,  and  hardened  in  50  parts  of  a  saturated  aqueous 
solution  of  mercuric  chloride  and  3  parts  of  formalin,  for  2  days.  It 
was  then  washed  in  flowing  water  for  12  hours,  and  decalcified  in 
20  parts  of  formic  acid,  3  parts  of  glacial  acetic  acid,  and  30  parts  of 
distilled  water.  It  was  again  washed  in  water,  dehydrated, 
embedded  in  paraffin,  and  cross-sectioned.  The  sections  were 
fixed  to  cover  glasses,  stained  in  hrematoxylin  and  eosin,  dehy- 
drated, and  cleared  in  oil  of  bergamot  and  xylol.  Mount  in  balsam. 

Study  under  high  power.  The  preparation  is  given  to 
demonstrate  the  structure  of  the  pulp.  Observe  the  loose 
connective  tissue,  consisting  of  a  few  fibrils  and  branched 
connective  tissue  cells.  A  layer  of  odontoblasts  bounds 
the  pulp.  These  cells  have  a  column-shaped  body ;  from 


—  84  — 

this  two  branches  are  given  off,  pulpal  processes  communi- 
cating with  the  branched  cells  of  the  pulp,  and  tubular 
processes  which  enter  the  dentinal  tubules.  Sketch  under 
high  power  a  portion  of  the  pulp  with  odontoblasts  and  a 
segment  of  the  adjacent  dentine. 

(c)  Developing  Tooth  Showing  the  Enamel  Organ. 

A  cat  embryo  about  an  inch  in  length  was  hardened  in 
mercuric  chloride.  The  head  was  removed  and  stained  in  Dela- 
field's  hsematoxylin,  embedded  in  paraffin,  and  cro^s  sections  of  the 
face  made.  Fix  and  mount  in  balsam. 

Study  first  under  low  power.  The  preparation  shows 
the  nasal  cavities,  separated  from  each  other  by  the  nasal 
septum,  and  separated  from  the  oral  cavity  by  the  roof  of 
the  mouth.  From  the  floor  of  the  mouth  there  projects  a 
broad  fold,  the  tongue.  In  the  developing  lower  jaw  will 
be  seen  the  enamel  organs,  a  little  to  the  side  of  the  tongue ; 
also,  at  a  point  just  above,  in  the  developing  upper  jaw. 
Study  one  of  the  enamel  organs  under  high  power.  It  has 
the  shape  of  a  shallow  bowl,  and  is  in  connection  with  the 
epithelium  lining  the  mouth  cavity  by  a  narrow  stalk. 
The  enamel  organ  is  developed  from  this  epithelium.  The 
embryonic  connective  tissue  under  the  enamel  organ  shows 
a  denser  structure,  and  much  more  cell  proliferation  than 
the  surrounding  connective  tissue;  it  constitutes  the 
dental  papilla.  Sketch  the  section  in  outline  as  seen 
under  low  power,  and  one  of  the  enamel  organs  and  dental 
papillae  as  seen  under  high  power. 

(d)  Developing    Tooth    Showing    Formation    of 

Dentine  and  Enamel. 

The  head  of  an  embryo  cat  (about  4  to  5  inches  long)  was 
hardened  in  picro-sulphuric  acid,  stained  in  Delafield's  hsematoxy- 
lin,  embedded  in  paraffin,  cross-sections  of  the  face  made,  fixed  to 
cover  glasses,  counter-stained  in  eosin,  dehydrated,  and  cleared  in 
oil  of  bergamot  and  xylol.  Mount  on  balsam. 

Study  first  under  low  power.  The  same  general 
structure  will  be  recognized  in  this  section  as  in  (c)  of  this 
lesson.  Study  one  of  the  developing  teeth  under  high 
power.  In  the  enamel  organ  three  kinds  of  cells  can  be 


—  85  — 

made  out.  The  lower  layer  consists  of  very  regular,  tall, 
columnar  cells,  the  enamel  cells.  These  cells  are  engaged 
in  depositing  the  enamel,  consisting  of  prisms  of  a  struct- 
ureless substance.  Above  the  enamel  cells  are  found  2  to 
3  layers  of  short  columnar  or  polyhedral  cells.  Then  there 
is  a  layer  of  varying  thickness  composed  of  highly  meta- 
morphosed epithelial  cells,  seen  in  the  form  of  flattened 
plates  or  fibres,  or  spindle-shaped  or  star-shaped  cells. 
This  layer  constitutes  the  enamel  pulp.  The  outer  layer 
is  composed  of  two  or  three  rows  of  flattened,  irregular 
cells  in  connection  with  the  epithelium  lining  the  mouth 
cavity. 

In  the  dental  papilla  the  cells  of  the  peripheral  part 
have  assumed  a  very  regular  arrangement,  and  are  colum- 
nar in  shape.  They  constitute  the  layer  of  odontoblasts, 
and  are  engaged  in  the  deposition  of  dentine.  The  dentine 
is  quite  deeply  stained  in  eosin  in  this  section.  In  it  fine 
canals  can  be  made  out,  into  which  the  processes  of  the 
odontoblasts  extend.  The  remaining  portion  of  the  dental 
papilla  shows  the  structure  of  embryonic  connective  tissue. 
Make  an  outline  sketch  of  the  section  as  shown  under  low 
power,  and  of  one  of  the  developing  teeth  as  seen  under 
high  power. 


DRAWINGS  FOR  LESSON  XX. 


DRAWINGS  FOR  LESSON  XX, 


DRAWINGS  FOR  LESSON  XX. 


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OF   THE 

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LESSON  XXI. 
DIGESTIVE  TRACT  (Concluded). 

(a)  (Esophagus. 

The  oesophagus  of  a  dog  was  hardened  in  alcohol,  stained  in 
borax  carmine,  embedded  in  paraffin,  and  cross-sectioned.  Fix  to 
the  slide,  and  mount  in  balsam. 

Study  first  under  low  power,  and  notice  the  stratified 
pavement  epithelium  resting  on  a  papillated  inucosa, 
which  is  limited  externally  by  a  muscularis  mucosa.  Then 
follows  the  submucosa,  a  fibrous  tissue  coat  containing  the 
larger  vessels.  Mucous  glands  may  be  seen  in  this 
stratum.  Next  is  a  muscular  coat  composed  of  inner  cir- 
cular and  outer  longitudinal  bundles.  If  the  section  is 
from  the  upper  third  of  the  oesophagus,  the  muscular  tissue 
is  largely  of  the  striped  variety,  below  this,  non-striped. 
Sketch  a  segment  of  the  wall  as  seen  under  low  power. 

(b)  Junction  of  the  (Esophagus  and  Cardiac  End 

of  the  Stomach. 

A  portion  of  the  alimentary  canal,  including  the  lower  end  of 
the  oesophagus  and  cardiac  end  of  the  fttOfftftafe,  was  removed, 
pinned  out  on  a  flat  cord,  and  hardened  in  absolute  alcohol.  A 
small  piece  including  the  oesophagus  and  cardiac  end  of  the  stomach 
was  cutout,  stained  in  borax  carmine,  embedded  in  paraffin,  and 
sectioned.  Fix  to  the  slide,  and  mount  in  balsam. 

Study  under  low  power.  The  cesophageal  portion  of 
the  section  shows  the  structure  seen  in  preparation  (a)  of 
this  lesson :  the  epithelial  lining  is  stratified  pavement 
Avhich  ends  abruptly  at  the  termination  of  the  oesophagus. 
Large  mucous  glands  are  usually  found  in  the  mucosa  and 
submucosa.  In  the  dog  a  layer  of  striped  muscle  fibres, 
which  extends  as  a  thin  sheath  outside  of  the  non-striped 


muscle  to  the  end  of  the  oesophagus,  passes  for  a  short  dis- 
tance on  to  the  wall  of  the  stomach.  The  portion  of  the 
section  coming  from  the  stomach  is  lined  by  a  single  layer 
of  columnar  cells.  A  few  imperfectly  formed  peptic  glands 
are  also  seen.  Sketch  the  portion  of  the  section  showing 
the  junction  of  the  stratified  pavement  and  the  single  layer 
of  columnar  epithelium  with  the  underlying  mucosa,  as 
seen  under  low  power. 

(c)  Cardiac  End  of  the  Stomach. 

Pieces  from  the  cardiac  end  of  the  stomach  of  a  dog  were 
hardened  in  absolute  alcohol,  embedded  in  paraffin,  and  sectioned. 
The  sections  were  fixed  to  cover  glasses,  and  stained  in  the  Ehrlich- 
Biondi  triple  stain,  dehydrated,  and  cleared  in  oil  of  bergamot  and 
xylol.  Mount  on  balsam. 

Study  first  under  low  power.  Observe  the  mucosa 
with  the  cardiac  glands,  the  muscularis  mucosa,  the  fibrous 
submucosa  with  the  larger  vessels,  the  muscular  coat  com- 
posed of  two  or  three  layers  of  non-striped  muscle,  and  the 
peritoneal  coat.  Under  high  power  the  finer  details  are 
mado  out.  The  peptic  glands  are  simple  or  branched 
tubular  glands,  with  short  neck  and  comparatively  long 
secreting  tubules.  In  the  latter  two  kinds  of  cells  are 
found,  the  central,  chief,  or  peptic  cells,  stained  a  faint 
green  color,  and  the  parietal  or  oxyntic  cells  of  Langley,  of 
oval  shape  and  stained  a  reddish  tinge. 

This  section,  and  the  sections  of  the  pyloric  end  of  the 
stomach  and  of  the  small  and  large  intestines  are  to  be 
sketched  side  by  side  in  the  spaces  outlined  for  you  on  two 
of  the  pages  reserved  for  the  drawings  of  this  lesson.  The 
sketches  are  to  be  so  made  that  the  muscularis  mucosa  of 
each  of  the  four  sections  falls  on  the  dotted  line  running 
across  the  pages.  The  relative  thicknesses  of  the  several 
coats  should  be  shown  as  accurately  as  possible.  Sketch 
under  low  power. 

(d)  Pyloric  End  of  the  Stomach. 

The  tissue  was  taken  from  the  pyloric  end  of  a  dog's  stomach, 
hardened  in  alcohol,  stained  in  borax  carmine,  and  embedded  in 
paraffin.  Mount  in  balsam. 


Observe  that  in  the  pyloric  glands  the  duct  is  longer, 
the  secreting  tubule  correspondingly  shorter,  and  lined  by 
only  one  kind  of  cells,  these  resembling  the  chief  cells  in 
the  cardiac  glands.  The  section  may  show  small  masses 
of  adenoid  tissue  in  the  deeper  portion  of  the  mucosa. 
Sketch  as  above  directed. 

(e)  Section  of   the  Pyloric   End  of  the   Stomach 

and   Small  Intestine,  Showing  Brunner's 
Glands. 

A  piece  of  the  intestinal  canal,  including  the  junction  of  the 
pyloric  end  of  the  stomach  and  the  sinaH  intestine,  was  removed 
from  a  cat,  pinned  out  on  a  flat  cork,  hardened  in  absolute  alcohol, 
stained  in  borax  carmine,  embedded  in  paraffin,  and  so  cut  as  to 
include  both  pyloric  end  and  small  intestine  in  the  section.  Fix  to 
the  slide,  and  mount  in  balsam. 

Study  under  low  power.  The  portion  of  the  section 
from  the  pyloric  end  will  show  the  structure  above 
described.  In  the  portion  from  the  small  intestine, 
observe  the  villi  and  the  crypts  of  Lieberkiihn  in  the 
mucosa,  and  Brunner's  glands  in  the  submucosa.  The 
latter  are  compound  tubular  glands  with  the  secreting 
tubules  in  the  submucosa.  They  are  grouped  about  the 
ends  of  the  long  ducts  which  pass  up  through  the  muscu- 
laris  mucosa  and  mucosa,  opening  in  the  depressions 
between  the  villi.  In  the  cat  the  secretion  of  these  glands 
is  mucous.  Sketch  a  portion  of  the  small  intestine  show- 
ing Brunner's  glands  as  seen  under  low  power. 

(f)  Small  Intestine. 

Small  pieces  from  the  small  intestine  of  a  dog  were  hardened 
in  mercuric  chloride,  stained  in  Delafield's  luematoxylin,  embedded 
in  paraffin,  and  cross-sectioned.  Fix  sections  to  the  slide,  and 
mount  in  balsam. 

Study  first  under  low  power.  Note  the  villi,  conical 
projections  of  the  mucosa,  covered  by  a  single  layer  of 
columnar  cells.  Observe  the  chyle  vessel  in  the  center  of 
each  villus.  In  the  mucosa  are  found  many  tubular 
glands,  the  crypts  of  Lieberkuhn,  coming  to  the  surface 
between  the  villi.  The  mucosa  is  bounded  below  by  a 
muscularis  mucosa  composed  of  two  or  three  layers  of  non- 


striped  muscle  cells.  Beneath  this  coat  is  a  loose  fibro- 
elastic  coat,  the  submucosa,  in  which  several  quite  large 
arteries  and  veins  will  be  seen  in  section.  The  muscle 
coat  is  composed  of  an  inner  circular  and  an  outer  longi- 
tudinal layer  of  non-striped  muscle  tissue.  The  serous 
coat  is  seen  as  a  thin  layer  of  fibrous  tissue,  covered  on  the 
outer  surface  by  a  layer  of  endothelial  cells.  Sketch  the 
section  by  the  side  of  the  drawing  of  the  pyloric  end  of  the 
stomach. 

(g)  Small  Intestine  Showing  Peyer's  Patch. 

A  piece  of  the  small  intestine  known  to  contain  a  Payer's  patch 
was  hardened  in  mercuric  chloride,  stained  in  Delafield's  hamiatoxy- 
lin,  embedded  in  paraffin,  and  sectioned.  Fix  and  mount  in 
balsam. 

Study  under  low  power.  Observe  the  several  nodules 
of  simple  adenoid  tissue  in  the  mucosa^and  submucosa, 
pushing  aside  the  crypts  of  Lieberkiihn,  and  often  coming 
up  under  the  epithelial  lining  of  the  intestine  and  causing 
an  elevation  of  the  epithelial  layer.  Sketch  a  portion  of 
the  intestine  showing  a  lymph  nodule  as  seen  under  low 
power. 

(h)  Injected  Small  Intestine. 

The  mesenteric  artery  of  a  c<4  was  injected  with  gelatine- 
carmine,  hardened  in  alcolml,  stained  .in  Delatield's  hsematoxylin, 
embedded  in  paraffin,  and  cross-sectioned.  Fix  and  mount  in 
balsam. 

Use  low  power.  Note  the  arrangement  of  the  in- 
jected vessels  in  the  muscular  coat,  in  the  submucosa, 
about  the  crypts  of  Lieberkiihn,  and  in  the  villi.  Sketch 
a  portion  of  the  wall  under  low  power. 

(i)  Large  Intestine. 

Pieces  of  the  large  intestine  of  a  dog  were  hardened  in  alcohol, 
stained  in  borax  carmine,  embedded  in  paraffin,  and  sectioned. 
Fix  to  the  slide,  and  mount  in  balsam. 

Notice  that  in  the  hirge  intestine  no  villi  are  found. 
Simple  tubular  glands,  the  crypts  of  Lieberkiihn,  are 
observed.  They  are  placed  vertically  in  the  mucosa,  and 
are  lined  by  a  single  layer  of  short  columnar  cells,  the 
great  majority  of  these  appearing  as  goblet  cells.  Sketch 
by  the  side  of  the  small  intestine  as  above  directed. 


—  93  - 
(j)  Pancreas. 

The  pancreas  of  a  dog  was  hardened  in  mercuric  chloride, 
stained  in  Dehifield's  hsematoxylin,  embedded  in  paraffin,  and  sec- 
tioned. Fix  sections  to  the  slide,  and  mount  in  balsam. 

Study  first  under  low  power.  The  structure  of  the 
pancreas  is  in  many  ways  similar  to  that  of  a,  serous  sali- 
vary gland.  Note  the  inner  granular  zone  in  the  cells 
lining  the  alveoli;  the  nucleus  is  found  in  the  outer  zone.' 
Sketch  several  alveoli  as  seen  under  high  power. 

DRAWINGS  FOR  LESSON  XXI. 


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DRAWINGS  FOR  LESSON  XXI. 


CARDIAC  END  OF  STOMACH. 


PYLORIC  END  OF  STOMACH. 


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DRAWINGS  FOR  LESSON  XXI. 


SMALL  INTESTINE. 


LARGE  INTESTINE. 


DRAWINGS  FOR  LESSON  XXI. 


—  97  — 


LESSON  XXII. 

LIVER,   TRACHEA,   LUNG,    AND    THYROID. 

(a)  Liver  Cells. 

Small  pieces  from  the  liver  of  a  cat  were  macerated  for  24  hours 
in  llanviers  alcohol,  and  for  2  hours  in  0.5%  osinic  acid.  Tease 
in  gum  glycerine. 

Study  under  high  power.  The  liver  cells  are  polyhe- 
dral in  form,  and  possess  a  distinct  intracellular  network. 
Fat  globules,  stained  black,  may  be  seen  in  the  cells.  As 
a  rule  a  single  spherical  nucleus  is  found.  Sketch  several 
as  seen  under  high  power. 

(b)  Injected  and  Stained  Liver. 

The  liver  of  a  pig  was  injected  through  the  portal  vein  with 
Berlin  blue,  hardened  in  alcohol,  stained  in  borax  carmine, 
embedded  in  paraffin,  and  sectioned.  Fix  and  mount  in  balsam. 

Study  under  high  power.  Note  that  the  gland  is  com- 
posed of  lobules,  and  injected  interlobular  branches  of  the 
portal  vein  are  seen  between  them.  From  these  interlobu- 
lar vessels  capillaries  pass  into  the  lobule  and  unite  in  an 
intralobular  vessel,  this  emptying  into  a  sublobular  vein. 
The  liver  cells  appear  arranged  in  columns  between  the 
capillaries.  Sketch  two  of  the  lobules,  showing  the  arrange- 
ment of  blood  vessels  and  liver  cells  as  seen  under  low 
power. 

(c)  Human  Liver. 

Small  pieces  of  a  human  liver  were  hardened  in  mercuric  chlo- 
ride, embedded  in  paraffin,  and  sectioned.  The  paraffin  was 
removed  from  the  sections  with  xylol ;  they  were  then  stained  in 
hsematoxylin  and  eosin,  dehydrated,  and  cleared  in  oil  of  berga- 
rnot.  Mount  in  balsam.  Study  first  under  low  power. 

In  the  human  liver  the  hepatic  lobules  are  separated 
one  from  the  other  by  only  a  very  small  amount  of  inter- 


—  98  — 

lobular  connective  tissue.  The  intralobular  vein  is  quite 
easily  made  out  in  the  center  of  each  lobule ;  from  this  the 
cords  of  liver  cells  radiate  in  the  form  of  irregular  anasto- 
mosing columns,  between  which  the  open  capillaries,  often 
containing  a  few  red  blood  cells  deeply  stained  in  eosin, 
can  be  seen.  Here  and  there  a  bile  duct,  lined  by  a  single 
la}^er  of  cubical  or  short  columnar  cells,  will  be  seen  in  the 
interlobular  connective  tissue.  Sketch  a  portion  of  the 
section,  including  a  bile  duct,  as  seen  under  high  power. 

(d)  Liver  with  Bile  Capillaries  Stained  According 

to  Oppel's  Method. 

Small  pieces  of  the  liver  were  hardened  in  a  solution  of  potas- 
sium bichromate  and  osmic  acid  (Ramon  y  Cajal)  for  three  days, 
then  transferred  to  a  %  %  solution  of  silver  nitrate,  in  which  they 
remained  for  several  days.  The  sections  are  in  turpentine  ;  mount 
in  hard  balsam. 

The  bile  capillaries  are  stained  black.  Two  sections 
are  given,  one  from  the  liver  of  an  embryo  rat,  in  this  the 
compound  tubular  character  of  the  gland  is  easily  made 
out ;  the  second  from  the  liver  of  a  young  kitten,  showing 
an  apparent  network  of  bile  capillaries  between  the  liver 
cells.  Sketch  a  portion  of  each  section  as  seen  under  high 
power. 

(e)  Trachea. 

Small  pieces  from  the  trachea  of  a  young  cat  were  hardened  in 
mercuric  chloride,  stained  in  Delafield's  haematoxvlin,  embedded 
in  paraffin,  and  sectioned.  Fix  and  mount  in  balsam. 

Study  under  high  power.  The  trachea  is  lined  by  strati- 
fied ciliated  columnar  epithelium  resting  on  a  mucosa  of 
loose  fibre-elastic  tissue,  in  the  outer  portion  of  which  the 
elastic  fibres  form  a  well-marked  layer.  The  fibrous  sub- 
mucosa  contains  the  mucous  tracheal  glands.  In  the 
external  fibrous  coat  is  found  the  hoop  of  hyaline  cartilage. 
Between  the  ends  of  the  cartilage  the  outer  layer  is  com- 
posed of  fibrous  and  non-striped  muscle  tissue.  Sketch  a 
segment  of  the  trachea  as  seen  under  high  power. 

(f)  Lung. 

The  lung  of  a  cat  was  distended  with  absolute  alcohol  before 
opening  the  chest  cavity ;  after  ligating  the  trachea  the  lung  was 


—  99  — 

carefully  removed,  and  suspended  for  several  days  in  alcohol,  then 
divided  into  small  pieces,  stained  in  borax  carmine  or  Delafield's 
luetnatoxylin,  embedded  in  paraffin,  and  sectioned.  Fix  to  the 
sl'de,  and  mount  in  balsam. 

Study  first  under  low  power,  and  notice  the  bronchi, 
blood  vessels,  and  alveoli.  The  sections  of  the  large 
bronchi  show  a  lining  of  stratified  ciliated  columnar  epithe- 
lium resting  on  a  fibrous  mucosa,  in  which  small  mucous 
glands  may  be  observed.  Next,  there  is  a  band  of  non- 
striped  muscle  tissue,  outside  of  which  cartilage  plates  may 
be  seen  in  cross-section.  The  small  bronchi  are  lined  by  a 
single  layer  of  ciliated  columnar  cells,  and  the  cartilaginous 
plates  are  wanting.  Try  and  make  out  the  capillaries 
between  the  alveoli ;  a  few  blood  cells  may  be  seen  in  them. 
Sketch  a  segment  from  the  wall  of  a  large  bronchus ;  also 
a  small  bronchiole  with  a  few  of  the  surrounding  alveoli,  as 
seen  under  high  power. 

(g)  Thyroid  Gland. 

A  piece  of  the  thyroid  gland  of  a  dog  was  hardened  in  mercuric 
chloride,  embedded  in  paraffin,  sectioned,  and  fixed  to  cover 
glasses.  The  sections  were  then  stained  in  hrematoxylin  and 
eosin,  dehydrated,  and  cleared  in  oil  of  bergainot  and  xylol.  Mount 
on  balsam.  , 

Study  first  under  low  power.  The  gland  is  surrounded 
by  a  fibrous  capsule.  Oval  or  round  alveoli,  lined  by  a 
single  layer  of  cubical  cells,  are  seen.  The  alveoli  are  filled 
with  a  substance  which  stains  deeply  in  eosin.  Sketch 
several  alveoli  under  high  power. 


DRAWINGS  FOR  LESSON  XXII. 


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—  io3— 


LESSON  XXIII. 
KIDNEY,  ADRENAL,  AND  PROSTATE. 

(a)  Isolated  Tubules  of  Kidney. 

Pieces  from  the  kidney  of  a  small  mammal  were  macerated  for 
24  hours  in  a  30  %  solution  of  hydrochloric  acid,  and  washed 
for  %  hour  in  flowing  water.  Tease  very  carefuily  in  gum  glycer- 
ine. 

Different  portions  of  the  tubules  will  be  seen  in  the 
field.  Sketch  a  Malpighian  corpuscle,  a  convoluted  tubule, 
and  a  loop  of  Henle,  under  low  power. 

(b)  Longitudinal  Section  of  a  Small  Kidney. 

The  kidney  of  a  young  rat  or  other  small  mammal  was  hardened 
in  mercuric  chloride,  stained  in  Delafield's  haematoxylin,  and  longi- 
tudinal sections  cut.  The  sections  were  fixed  to  cover  glasses  and 
counter-stained  in  eosin,  dehydrated,  and  cleared  in  oil  of  bergamot 
and  xylol.  Mount  on  balsam. 

Study  first  under  low  power.  In  this  preparation  only 
one  Malpighian  pyramid  is  seen.  In  the  medullary  por- 
tion the  tubules  have  a  more  or  less  straight  direction, 
radiating  from  the  apex  toward  the  base  of  the  Malpighian 
pvramid.  In  the  cortex  are  observed  bundles  of  straight 
collecting  tubules  arranged  in  the  form  of  pyramids  (the 
pyramids  of  Ferrein  or  medullary  rays),  the  bases  of  which 
rest  on  the  base  of  the  Malpighian  pyramid.  Between  the 
medullary  rays  is  found  the  labyrinth  of  the  kidney,  com- 
posed of  Malpighian  corpuscles,  proximal  and  distal  convo- 
luted tubules,  and  spiral  and  zigzag  portions  of  the  urin- 
iferous  tubules.  Study  the  form  and  structure  of  the 
epithelium  lining  the  different  portions  of  the  uriniferous 
tubules  under  high  power.  Make  an  outline  sketch  of  the 
section  as  seen  under  low  power. 


— 104 — 

(c)  Injected  Kidney. 

The  kidney  of  a  dog  was  injected  with  Berlin  blue  through  the 
renal  artery,  hardened  in  alcohol,  stained  in  borax  carmine,  and 
embedded  in  paraffin.  Fix  and  mount  in  balsam. 

Study  under  low  power,  observing  the  injected  inter- 
lobular  arteries.  Note  the  glomeruli  with  their  afferent 
and  efferent  vessels,  the  capillary  network  about  the  convo- 
luted tubules,  and  the  straight  capillaries  of  the  medulla. 
Sketch  a  portion  of  the  cortex  and  medulla  seen  under  low 
power. 

(d)  Human  Kidney. 

A  human  kidney  was  cut  transversely  into  flat  pieces  about  }£ 
of  an  inch  thick,  hardened  in  mercuric  chloride,  embedded  in  par- 
affin, and  sectioned.  The  sections  were  fixed  to  cover  glasses,  stained 
in  hsematoxylin  and  eosin,  dehydrated,  and  cleared  in  oil  of  her- 
gamot  and  xylol.  Mount  on  balsam. 

Study  under  low  power.  Observe  the  relatively  thick 
capsule  of  fibrous  tissue,  and  a  narrow  zone  of  convoluted 
tubules  just  below  the  capsule,  in  which  no  Malpighiaii 
corpuscles  are  found.  Locate  the  medullary  rays  of  the 
cortex;  between  these  are  the  portions  of  the  uriniferous 
tubules  constituting  the  labyrinth  of  the  kidney.  Note  the 
scarcity  of  the  connective  tissue  between  the  tubules ;  also 
the  interlobular  vessels  passing  up  between  the  medullary 
rays.  In  the  medullary  portion  the  collecting  tubules  con- 
verge toward  the  apex  of  the  Malpighian  pyramid.  The 
largest  ones  near  the  apex  of  the  pyramid  are  known  as  the 
tubules  of  Bellini.  Between  the  straight  collecting  tubules, 
capillaries  often  filled  with  blood  are  to  be  looked  for. 
Sketch  a  portion  of  the  cortex  and  of  the  medulla  as  seen 
under  high  power,  reproducing  as  accurately  as  possible 
the  character  of  the  epithelium  lining  the  different  parts  of 
the  uriniferous  tubules. 

(e)  Suprarenal  Body. 

The  suprarenal  body  of  a  guinea  pig  was  hardened  in  mercuric 
chloride,  stained  in  Delafield's  hsematoxylin,  embedded  in  par- 
affin, and  sectioned.  Fix  and  mount  in  balsam. 

Study  first  under  low  power.  Observe  the  fibrous 
capsule  from  which  septa  pass  into  the  gland.  The  gland 


—io5— 

tissue  is  arranged  in  an  outer  more  deeply  stained  border, 
the  cortical  zone,  and  a  central  portion,  the  medulla.  In 
the  cortex  the  granular  cells  are  arranged  in  small  groups 
in  its  peripheral  portion;  this  is  called  the  glomerular  zone. 
Then  comes  a  broad  area,  in  which  the  cells  are  arranged 
in  quite  regular  columns,  the  fascicular  zone.  Finally 
there  is  a  third  stratum  next  to  the  medulla  in  which 
anastomosing  strands  of  cells  are  made  out ;  this  is  the 
reticular  zone.  In  the  medulla  are  to-be  seen  cords  of 
irregular,  pigmented  cells,  between  which  large  vascular 
spaces  are  found.  Sketch  a  band  through  the  cortex  and 
medulla  as  seen  under  high  power. 

(f)  Prostate. 

The  prostate  of  a  dog  was  divided  transversely  into  several 
pieces,  hardened  in  mercuric  chloride,  stained  in  Delafield's  heema- 
toxylin,  and  sectioned.  The  sections  were  fixed  to  cover  glasses, 
and  counter-stained  in  eosin,  dehydrated,  and  cleared  in  oil  of  ber- 
gamot  and  xylol.  Mount  on  balsam. 

Study  first  under  low  power.  The  capsule  of  the  pros- 
tate is  made  up  largely  of  non-striped  muscle  tissue  and  a 
small  amount  of  fibrous  tissue.  From  the  capsule  fibro- 
muscular  trabeculse  pass  toward  the  urethral  canal,  form- 
ing a  framework  for  the  glandular  structure.  The  glands 
are  compound  tubular.  The  secreting  tubules  are  lined  by  a 
single  layer  of  short  columnar  cells.  The  gland  ducts 
which  empty  into  the  urethra  are  relatively  large,  and 
lined  for  a  distance  with  transitional  epithelium.  In  the 
outer  portion  of  the  prostate,  and  here  and  there  in  the 
trabeculse  between  the  glands,  smaller  or  larger  sympa- 
thetic ganglia  are  to  be  seen.  The  prostatic  urethra  is  lined 
by  transitional  epithelium.  Sketch  about  i  of  the  section 
as  seen  under  low  power,  and  a  portion  of  one  of  the  glands 
as  seen  under  high  power. 


DRAWINGS  FOR  LESSON  XXIII. 


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LESSON  XXIV. 

MALE  AND  FEMALE  ORGANS  OF  REPRO- 
DUCTION. 

(a)  Testis. 

The  testis  of  a  dog  was  divided  transversely  into  thin  pieces, 
hardened  in  mercuric  chloride,  stained  in  Delafield's  hrematoxylin, 
embedded  in  paraffin,  and  sectioned.  Fix  to  slide,  and  mount  in 
balsam. 

Use  the  low  power.  The  section  is  given  to  show  the 
general  structure  of  the  gland.  Observe  the  fibrous  tissue 
capsule,  the  tunica  albuginea,  surrounding  the  gland. 
From  this  septa  pass  into  the  parenchyma,  and  form  a 
framework  for  the  seminiferous  tubules.  These  are  seen 
cut  in  cross  and  oblique  section.  They  are  lined  by  several 
layers  of  epithelial  cells.  Some  are  filled  with  spermatozoa. 
Off  to  one  side  of  the  testis  is  seen  the  epididymis.  The 
round  or  oval  twisted  tube  as  seen  in  cross  section  is  lined 
by  a  layer  of  stratified  ciliated  columnar  epithelium.  The 
wall  is  composed  of  a  fibrous  mucosa ;  outside  of  this  is  a 
layer  of  non-striped  muscle  tissue.  Near  the  epididymis  is 
the  vas  deferens.  It  shows  a  relatively  broad  outer  wall 
of  non-striped  muscle  tissue,  the  majority  of  the  cells  of 
which  are  arranged  transversely.  The  narrow  fibrous 
mucosa  is  lined  by  stratified  ciliated  columnar  epithelium. 

Sketch  a  portion  of  the  testis  under  low  power,  and  a 
tubule  of  the  epididymis  and  the  vas  deferens  as  seen  under 
high  power. 

(b)  Section   of  the  Testis    Showing    Spermato- 

genesis. 

Very  small  pieces  of  a  guinea  pig's  testis  were  hardened  for  24 
hours  in  a  solution  composed  of  two  parts  of  2  \%  osmic  acid, 
three  parts  of  2  %  platinum  chloride,  and  five  parts  of  a 
saturated  aqueous  solution  of  mercuric  chloride.  They  were  then 


washed  for  a  few  hours  in  flowing  water,  embedded  in  paraffin, 
sectioned,  fixed  to  cover  glasses,  stained  in  Heidenhain's  iron-lack- 
haematoxylin,  counter-stained  in  a  saturated  aqueous  solution  of 
acid  fuchsin,  washed,  dehydrated,  and  cleared  in  oil  of  bergamot 
and  xylol.  Mount  on  balsam. 

Study  under  high  power.  On  moving  the  section 
about  you  will  observe  that  the  variously  cut  seminiferous 
tubules  show  different  stages  of  development.  Within  the 
tunica  propria  of  a  resting  tubule  are  seen  several  layers  of 
cells,  in  the  outermost  of  which  are  found  the  spermat- 
gones  (parent  cells),  and  the  lower  portion  of  the  support- 
ing cells  of  Sertoli.  Then  comes  a  layer  of  quite  large 
cells,  the  spermatocytes  (mother  cells),  descendants  of  the 
spermatogones.  Lining  the  lumen  of  the  tubules  are  found 
several  layers  of  small  cells,  the  spermatoblasts  (daughter 
cells).  The  mother  cells  dividing  by  indirect  cell  division 
give  rise  to  the  small  inner  cells;  from  these  the  sperma- 
tozoa are  developed.  Search  for  a  tubule  showing  the 
fusion  of  the  spermatoblasts  with  the  supporting  cells  of 
Sertoli.  Other  tubules  will  show  the  successive  steps  in 
the  development  of  the  spermatozoa  in  these  cells  until 
they  are  found  free  in  the  lumen  of  the  tubule.  Observe 
especially  the  change  undergone  by  the  accessory  nucleus 
during  the  development  of  the  spermatoblasts  into  the  sper- 
matozoa. In  the  spermatoblast  the  accessory  nucleus  is 
seen  as  a  relative  large  ball  stained  black  in  this  prepara- 
tion ;  it  is  situated  on  one  side  of  the  nucleus.  As  develop- 
ment begins  the  accessory  nucleus  becomes  demilunar  in 
shape  and  applies  itself  to  the  nucleus.  It  then  assumes  a 
triangular  shape,  and  finally  becomes  the  head  cap  of  the 
fully  developed  spermatozoa.  Sketch  portions  of  3  or  4 
tubules  showing  the  several  stages  of  spermatogenesis,  as 
seen  under  high  power. 

(c)  Ovary. 

The  ovary  of  a  rabbit  or  cat  was  hardened  in  mercuric  chloride, 
stained  in  Delafield's  hsematoxylin,  embedded  in  paraffin,  and 
sectioned.  Fix  and  mount  in  balsam. 

Study  first  under  low  power.  The  ovary  is  covered  by 
a  single  layer  of  germinal  epithelium.  It  consists  of  a 
medullary  portion,  in  which  large  vessels  are  found,  sur- 


rounded  by  a  cortical  portion  containing  Graafian  follicles 
seen  in  different  stages  of  development.  The  framework  or 
stroma  is  composed  of  fibrous  and  non-striped  muscle  tissue. 
A  fully  developed  Graafian  follicle  consists  of  a  dense  sur- 
rounding stroma;  it  is  lined  by  the  mem  bran  a  granulosa 
composed  of  several  layers*  of  small  cells;  in  one  portion  of 
the  follicle  this  lining  is  thickened  to  form  the  discus  pro- 
ligerus  ;  •  in  this  discus  the  ovum  is  embedded.  The  cavity 
of  the  follicle  is  filled  by  the  liquor  folliculi.  Other  follicles 
in  different  stages  of  development  will  also'be  observed;  in 
some  the  ovum  is  surrounded  by  only  a  single  layer  of 
cells;  in  others  by  several  layers,  while  in  still  others  the 
cavity  of  the  follicle  may  just  be  forming.  The  section 
may  show  a  portion  of  a  corpus  luteum.  If  this  is  well 
developed,  it  will  consist  of  a  mass  of  quite  large  cells  with 
relatively  small  nuclei,  between  which  a  network  ofcnpilla- 
ries  will  be  seen.  Sketch  several  Graafian  follicles  showing 
different  stages  of  development,  and  a  portion  of  the  corpus 
luteum  if  present,  as  seen  under  high  power. 

(d)  Uterus. 

A  human  uterus  was  divided  into  small  pieces,  hardened  in 
mercuric  chloride,  stained  in  Delafield's  haematoxylin,  embedded  in 
paraffin,  and  cross  sections  of  the  wall  made.  Fix  to  the  slide,  and 
mount  in  balsam. 

Study  first  under  low  power.  The  uterus  will  show 
mucous,  muscular,  and  serous  coats.  The  mucosa  is  com- 
posed of  loose  fibrous  tissue,  and  is  covered  by  a  layer  of 
ciliated  columnar  cells.  In  it  are  found  the  uterine  glands, 
simple  or  branched  tubules  lined  by  a  layer  of  ciliated 
columnar  cells.  The  next  coat  is  composed  of  non-striped 
muscle  tissue.  Fibrous  tissue  is  found  between  the  muscle 
bundles,  also  relatively  large  vessels.  Sketch  a  portion  of 
the  wall  as  seen  under  low  power. 

(e)  Vagina. 

The  vagina  of  a  dog  was  hardened  in  absolute  alcohol,  stained 
in  borax  carmine,  embedded  in  paraffin,  and  sectioned.  Fix  to  the 
slide,  and  mount  in  balsam. 

Use  the  high  power.  The  vagina  has  three  coats,  an 
inner  mucous,  composed  of  stratified  pavement  epithelium 


112 

and  a  fibrous  mucosa ;  next  is  the  muscular  coat,  composed 
of  an  inner  circular,  and  an  outer  longitudinal  layer  of 
non-striped  muscle  cells;  lastly  an  outer  fibro-elastic 
coat.  Sketch  under  high  power. 

(f)  Mammary  Gland. 

Small  pieces  taken  from  the  mammary  gland  of  a  nursing  bitch 
were  hardened  in  mercuric  chloride,  stained  in  Delafield's  haema- 
toxylin,  embedded  in  paraffin,  and  sectioned.  The  sections  were 
fixed  to  cover  glasses,  counter-stained  in  eosin,  dehydrated,  and 
cleared  in  oil  of  bergamot  and  xylol.  Mount  on  Balsam. 

Study  first  under  low  power.  Observe  the  acini  of  the 
gland  united  into  small  lobules  by  fibrous  tissue.  Under 
high  power  it  will  be  seen  that  the  acini  are  limited  by  a 
membrana  propria,  and  lined  by  a  single  layer  of  cubical  or 
polyhedral  cells.  In  the  protoplasm  of  these  cells,  fat 
droplets,  varying  in  size,  are  often  seen.  The  nucleus  is  in 
the  outer  part  of  the  cell.  The  lumen  of  the  acinus  is 
quite  large,  and  often  contains  granules,  droplets  of  fat,  and 
fragments  of  epithelium.  Sketch  a  number  of  acini  under 
high  power. 

DRAWINGS  FOR  LESSON  XXIV. 


DRAWINGS  FOR  LESSON  XXIV. 


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DRAWINGS  FOR  LESSON  XXIV. 


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LESSON  XXV. 

EYE,  EAR,  AND  OLFACTORY  MUCOUS 
MEMBRANE. 

(a)  Section  of  the  Anterior  Quadrant  of  the  Eye- 
ball. 

The  anterior  half  of  a  human  eye  was  hardened  in  Miiller's 
fluid.  After  hardening,  the  lens  was  removed,  and  a  quadrant 
including  a  portion  of  the  cornea  and  sclerotic  and  ciliary  region  cut 
out,  stained  in  Dplafield's  hsematoxylin,  embedded  in  paraffin, 
and  sectioned.  Fix  and  mount  in  balsam. 

Study  first  under  low  power.  The  cornea  is  composed 
of  5  layers,  named  in  order  from  before  backward,  as 
follows: — 

(1)  Stratified  pavement  epithelium. 

(2)  Bowman's   layer,    or   the    anterior  homogeneous 
lamella. 

(3)  Substantia  propria,  the  thickest  of    the    several 
coats,  composed  of  bundles  of  white  fibrous  tissue  arranged 
in  layers,  between  which  the  corneal  corpuscles  are  found. 

(4)  The  posterior  elastic  or  Descemet's  membrane. 

(5)  The  endothelium  of  the  anterior  chamber. 

In  the  sclera  the  bundles  of  white  fibrous  tissue  are 
densely  woven,  and  are  continuous  with  the  fibrous  tissue 
bundles  of  the  substantia  propria  of  the  cornea,  but  are  not 
so  regularly  arranged.  Observe  the  canal  of  Schlemm  as 
seen  in  cross-section  in  the  sclero-corneal  junction.  Of  the 
middle  layer,  the  iris,  ciliary  body,  and  anterior  portion  of 
the  choroid  are  included  in  this  section.  The  iris  is 
covered  anteriorly  by  a  layer  of  cells  continuous  with  the 
ones  found  on  the  posterior  surface  of  the  cornea.  The 
stroma  is  a  loose  fibrous  tissue  in  which  pigmented  and  un- 
pigmented  branched  cells  and  many  vessels  are  found.  The 
iris  is  covered  posteriorly  by  a  double  layer  of  deeply  pig- 


— u6— 

merited  cells,  the  pars  iridis  retinae.  The  fibres  of  the 
sphincter  of  the  iris  are  seen  in  cross-section  near  its  free  edge 
and  posterior  surface.  Observe  the  ligamentum  pectinatum, 
composed  of  trabeculae  of  fibrous  tissue  uniting  the  ciliary 
body  to  the  outer  coat  at  the -sclero-corneal  junction.  The 
spaces  between  the  trabeculae  of  this  ligament  communi- 
cate with  the  anterior  chamber,  and  are  known  as  Fon- 
tana's  spaces. 

The  ciliary  body  is  a  very  much  thickened  portion  of 
the  middle  coat;  it  is  continuous  anteriorly  with  the  iris, 
and  posteriorly  with  the  choroid.  It  is  composed  of  merid- 
ionally  placed  folds,  the  ciliary  processes,  and  the  ciliary 
muscle.  The  meridional  fibres  of  this  muscle  have  their 
origin  from  the  wall  of  the  canal  of  Schlemm  and  the 
adjacent  fibrous  tissue,  extending  backward  to  the  anterior 
portion  of  the  choroid  (the  tensor  choroidae),  and  into  the 
ciliary  body.  The  equatorial  or  circular  fibres  of  Miiller 
are  observed  in  cross  section  near  the  base  of  the  iris.  The 
ciliary  body  and  processes  are  covered  by  a  double  layer  of 
pigmented  cells,  the  pars  ciliaris  retinae.  Only  a  small 
portion  of  the  choroid  is  seen  in  this  section.  Note  how 
vascular  it  is.  In  the  stroma  many  branched  and  pig- 
mented connective  tissue  cells  are  seen. 

Notice  how  the  coats  of  the  retina  are  quite  abruptly 
reduced  to  a  double  layer  of  cells  at  the  ora  serata,  contin- 
uing over  the  ciliary  body  and  posterior  surface  of  the 
iris  as  the  pars  ciliaris  retinae  and  pars  iridis  retinae. 
Observe  the  suspensory  ligament  of  the  lens,  composed  of 
homogeneous  fibres,  these  seemingly  arising  from  the 
apices  of  the  ciliary  processes,  and  passing  from  these  to 
the  equator  of  the  lens,  some  uniting  with  the  capsule  of 
the  lens  on  its  anterior,  others  on  its  posterior  surface. 

Draw  this  preparation  as  seen  under  low  power. 


(b)  Lens  Fibres. 

A  lens  was   macerated  for  several  days    in  )^  %  solution   of 
hydrochloric  acid.     Tease  and  mount  in  gum  glycerine. 

Examine   under   high  power.     Sketch  several  of  the 
fibres,  as  seen  under  this  power. 


(c)  Retina. 

The  posterior  half  of  a  human  eye  was  hardened  in  mercuric 
chloride.  Small  pieces  of  the  retina  were  removed,  stained  in  Dela- 
neld'H  heematoxylin,  embedded  in  paraffin,  and  sectioned.  Fix  to 
the  slide,  and  mount  in  balsam. 

Use  high  power.  The  following  layers  are  made  out, 
named  in  order  from  before  backward  : — 

(1)  Internal  limiting  membrane. 

(2)  Layer  of  nerve  fibres. 

(3)  Layer  of  ganglion  cells. 

(4)  Inner  granular  or  molecular  layer. 

(5)  Inner  nuclear  layer. 

(6)  Outer  granular  or  molecular  layer. 

(7)  Outer  nuclear  layer. 

(8)  Outer  limiting  membrane  . 

(9)  Layer  of  rods  and  cones. 

(10)  Layer  of  pigment  cells;  this  layer  often  remains 
attached  to  the  choroid,  and  may  therefore  not  be  seen  in 
your  section. 

Sketch  under  high  power. 

(d)  Optic  Papilla. 

The  posterior  part  of  a  human  eye  was  hardened  in  Miiller's 
•fluid.  The  optic  papilla  with  a  small  portion  of  the  posterior  wall  and 
entering  optic  nerve  was  removed,  stained  in  Delafield's  haematoxy- 
lin,  embedded  in  paraffin,  and  sectioned.  Fix  and  mount  in  balsam. 

Study  first  under  low  power.  Note  the  dural  and  arach- 
noidal  sheaths  of  the  optic  nerve.  They  are  continuous 
with  the  sclerotic.  The  optic  nerve  fibres  passing  through 
the  lamina  cribrosa  and  bending  over  and  spreading  out  to 
form  the  layer  of  nerve  fibres  of  the  retina,  are  to  be  observed. 
Notice  the  excavation  in  the  optic  papilla.  The  nerve 
fibres  of  the  retina  are  on  the  anterior  surface ;  the  rods 
and  cones  point  toward  the  sclerotic.  The  section  may 
show  the  artery  centralis  retinae  in  longitudinal,  and  'some 
of  its  branches  in  cross  or  oblique,  section.  Sketch  under 
low  power. 

(e)  Cochlea  of  a  Guinea  Pig. 

The  cochlea  of  a  guinea  pig  was  hardened  in  Flemming's  solu- 
tion, decalcified  in  1  %  chromic  acid,  ami  embedded  in  celloidiu. 

. 

^  ^ 


— n8— 

The  sections  were  cut  in  a  direction  parallel  to  the  long  axis,  stained 
in  hsematoxylin  and  acid  fuchsin,  and  are  now  in  oil  of  bergamot. 
Mount  in  balsam. 

The  cochlear  canals  were  opened  on  one  side  so  that 
the  hardening  fluid  might  penetrate  more  easily.  Study 
first  under  low  power.  Observe  the  bony  axis  or  modeolus 
about  which  the  cochlear  canal  is  spirally  wound.  Sections 
of  it  are  seen  on  either  side  of  the  modeolus.  The  coch- 
lear canal  is  divided  into  two  portions  by  the  lamina 
spiralis,  a  bony  crest  attached  to  the  modeolar  wall  of  the 
canal,  and  the  basilar  membrane  extending  from  the  lamina 
spiralis  to  the  ligamentum  spiralis.  The  upper  portion  of 
the  canal  is  the  scala  vestibuli,  the  lower  the  scala  tympani ; 
they  are  lined  by  endothelial  cells.  A  triangular  canal,  the 
cochlear  duct  or  scala  media,  is  cut  off  from  the  scala  vesti- 
buli by  Reissner's  membrane.  In  the  cochlear  duct  resting 
on  the  basilar  membrane  is. found  the  organ  of  Corti,  in 
which  the  following  parts  are  to  be  distinguished :  The 
pillars  of  Corti,  arranged  in  the  form  of  an  arch  ;  the  inner 
and  the  three  or  four  outer  hair  cells;  Deiter's  cells  sup- 
porting the  outer  hair  cells ;  peripheral  to  the  organ  of 
Corti  are  Hensen's  cells,  in  these  fat  granules  are  found  in- 
the  guinea  pig.  Note  the  membrana  tectoria  resting  on 
the  organ  of  Corti.  Observe  the  spiral  ganglion  in  the 
lamina  spiralis;  from  this  nerve  fibres  pass  toward  the 
organ  of  Corti,  and  make  connection  with  the  hair  cells. 
They  may  be  seen  passing  through  the  tunnel  of  Corti. 

Sketch  under  low  power  the  entire  section,  also  under 
high  power  a  cochlear  duct  with  the  organ  of  Corti. 

(f)  Olfactory  Membrane  (Teased). 

The  olfactory  membrane  of  a  dog  was  fixed  and  macerated  in 
YJ,  % 'osmlc  acid.  Place  a  small  piece  of  the  macerated  tissue 
on  a  slide  and  cover  with  a  few  drops  of  methylene  blue,  allow  it  to 
stain  for  several  minutes,  then  wash  away  the  excess  of  stain  with 
distilled  water,  add  a  drop  of  gum  glycerine,  tease,  and  mount. 

Study  under  high  power,  and  search  for  olfactory  and 
sustentacular  cells.  Sketch  a  number  of  the  cells  as  seen 
under  this  power. 


(g)  Olfactory  Membrane  (Section). 

The  mucous  membrane  was  removed  from  the  septum  of  a 
rabbit's  nose,  hardened  in  mercuric  chloride,  stained  in  Delafield's 
hrematoxylin,  embedded  in  paraffin,  and  sectioned.  Fix  to  the 
slide,  and  mount  in  balsam. 

This  preparation  shows  olfactory  and  respiratory 
mucous  membrane.  The  epithelium  covering  the  latter  is 
stratified  ciliated  columnar.  In  the  regio  olfactoria  the 
two  kinds  of  cells  studied  in  the  teased  preparation  will  be 
observed.  In  the  mucosa,  which  is  composed  of  loose 
fibrous  tissue,  Bowman's  glands  will  be  seen  in  section. 
Sketch  under  high  power. 


DRAWINGS  FOR  LESSON  XXV. 

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DRAWINGS  FOR  LESSON  XXV. 


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DRAWINGS  FOR  LESSON  XXV. 


METHODS  FOR  LABORATORY  WORK. 


Methods  for  Macerating  Fresh  Tissues. 


Ranvier's  Alcohol. 

Place  small  pieces  of  the  tissue  to  be  macerated  in  33 
%  alcohol,  in  which  they  remain  from  12  to  24  hours;  then 
transfer  them  to  a  0.25  %  osmic  acid  solution  for  two  to 
four  hours.  They  can  -HOW  be  teased. 

This  method  is  very  useful  for  macerating  epithelial 
tissue ;  for  instance,  the  cells  lining  the  intestinal  canal  or 
the  trachea,  for  isolating  the  cells  of  the  liver,  etc. 

Caustic  Potash  Solution. 

Make  a  30  %  aqueous  solution  of  KOH.  Small 
pieces  are  placed  in  this  solution  for  15  to  20  minutes.  The 
maceration  is  then  interrupted  by  transferring  the  tissue 
to  a  saturated  aqueous  solution  of  acetate  of  potash  (it 
takes  about  60  parts  of  the  acetate  of  potash  to  saturate  40 
parts  of  water),  to  which  a  few  drops  of  glacial  acetic  acid 
have  been  added  (the  author  adds  five  to  six  drops  to  25 
c.  c.  of  the  saturated  solution).  In  about  30  minutes  the 
tissue  is  ready  for  teasing;  it  can,  however,  be  kept  along 
time,  several  months,  in  the  acetate  of  potash. 

This  method  is  used  for  macerating  non-striped  and 
heart  muscle,  also  epithelial  cells. 

Hydrochloric  Acid. 

A  30  %  aqueous  solution  is  used;  in  it  the  tis- 
sues remain  for  12  to  24  hours,  and  are  then  washed  in 
flowing  water  for  half  an  hour,  when  they  are  ready  to  be 
teased.  HCL  is  especially  useful  for  isolating  the  tubules 
of  the  kidney. 


—  126— 

Nitric  Acid  (Gage). 

A  30  %  aqueous  solution  is  used ;  small  pieces  of  the 
tissue  remain  in  the  fluid  about  24  hours,  and  are  then 
thoroughly  washed  in  water. 

Striped  muscle  fibres  are  well  macerated  by  this 
method. 

Sulphuric  Acid  (M.  Schultz  and  Ranvier). 

Place  the  tissue  to  be  macerated  on  the  slide,  add  a 
few  drops  of  strong  sulphuric  acid,  and  cover  with  a 
cover  glass.  In  a  few  moments  the  cells  can  be  separated 
by  gently  tapping  or  pressing  with  a  needle  the  top  of  the 
cover  glass. 

Useful  for  maceratingfhair,  nail,  and  horny  epidermis. 

Schultze's  Mixture. 

One  or  two  grams  of  crystals  of  chlorate  of  potash  are 
mixed  with  a.little  HN03,  only  enough  acid  being  used  to 
make  a  thick  paste.  In  this  the  tissue  to  be  macerated  is 
embedded  for  two  or  three  hours,  at  the  end  of  which  time 
the  tissue  may  be  removed  to  a  slide  and  teased.  If  it 
does  not  tease  easily,  embed  again  in  the  paste,  repeating 
at  intervals  of  about  thirty  minutes,  until  it  teases  readily. 

This  method  brings  out  nicely  the  branched  muscle 
fibres.  A  frog's  tongue  may  be  used. 

Osmic  Acid. 

A  1.0  °/o  aqueous  solution  of  osmic  acid  is  very  use- 
ful for  macerating  retina,  olfactory  membrane,  etc.  The 
tissues  remain  in  the  fluid  from  24  to  48  hours,  and  may 
then  be  teased. 

Macerated  tissues  are  to  be  teased  and  mounted  in 
glycerine,  glycerine  gelatin,  or  gum  glycerine. 

The  glycerine  gelatin  may  be  prepared  after  Fol's  for- 
mula : 

Water, 42  c.  c. 

Glycerine,  .         .         .••.'.         .        38  c.  c. 

Gelatin, 7  grms. 

Carbolic  acid, 1  grm. 

Cut  the  gelatin  in  small  pieces,  place  the  small  pieces 
into  a  clean  beaker,  and  add  the  water.  Warm  over  a 


—  127  — 

water-bath  until  the  gelatin  melts,  then  add  the  glycerine 
while  stirring  with  a  clean  glass  rod,  and  finally  add  the 
carbolic  acid.  Filter  through  a  hot  flannel. 

The  gum  glycerine  may  be  prepared  according  to  Far- 
rant's  formula : 

Glycerine,  .         .         .         .         .  50  c.  c. 

Water, 50  c.  c. 

Gum  Arabic  (powder),        ...  50  grms. 

Arsenious  acid,  ....  1  grm. 

Dissolve  the  arsenious  acid  in  the  water.  Place  the 
gum  arabic  in  a  glass  mortar  and  mix  it  with  the  water, 
then  add  the  glycerine.  Filter  through  a  wet  filter  paper 
or  through  fine  muslin. 


Methods  for  Hardening  and  Fixing  Tissues. 


Alcohol. 

When  possible  to  cut  the  tissue  into  small  pieces 
about  one  quarter  to  one-half  inch  cube,  it  is  best  to  place 
them  at  once  into  95%  alcohol;  here  they  remain 
for  four  or  five  days,  and  are  then  transferred  to  absolute 
alcohol  for  24  to  48  hours.  It  is  always  well  to  place  a 
layer  of  absorbent  cotton  in  the  hardening  jar  to  prevent 
the  tissues  from  resting  on  the  bottom.  The  fluid  can 
then  more  readily  touch  all  surfaces  of  the  tissue  to  be 
hardened.  Gland  tissue,  such  as  salivary  glands  and 
pancreas,  also  the  intestinal  canal,  are  well  hardened  by  this 
method.  Kahlden  states  that  this  method  is  especially 
useful  when  the  tissues  are  to  be  examined  for  bacteria. 

Bichloride  of  Mercury  Solution. 

Make  a  saturated  aqueous  solution  of  bichloride  of 
mercury.  This  is  most  easily  done  by  dissolving  80  to  100 
grins,  of  the  salt  in  a  liter  of  hot  distilled  water,  and  setting 
this  supersaturated  solution  aside  to  cool.  The  excess  will 
then  precpitate  out,  and  the  cold  saturated  solution  can 
then  be  decanted. 

Only  small  or  thin  pieces,  not  thicker  than  -Jth  of  an 
inch  should  be  placed  into  this  hardening  fluid.  They 
remain  in  the  solution  for  12  to  24  hours,  are  then 
thoroughly  washed  in  flowing  water  for  12  to  24  hours, 
transferred  .to  70%  alcohol  for  24  hours,  and  95%  alcohol 
for  another  day.  This  is  one  of  the  very  best  of  harden- 
ing fluids,  and  can  be  used  to  advantage  for  hardening  all 
glands,  mucous  membranes,  muscle,  etc.  It  is  especially 
useful  for  hardening  pathological  tissues.  Almost  any 
staining  fluid  may  be  used  after  it. 


-i29— 

Mercuric  Chloride  and  Formalin. 

The  author  has  used  to  good  advantage  the  following 
solution : 

Saturated  aqueous  solution  of  bichloride  of  mercury  100  parts 
Formalin 4  parts. 

In  this  solution  the  tissues  remain  for  24  to  48  hours, 
are  then  washed  in  flowing  water  for  24  hours,  and  the 
hardening  is  completed  in  70%  (24  hours.)  and  95% 
alcohol.  It  is  useful  for  hardening  teeth,  developing  teeth, 
developing  bone,  etc. 

Mercuric  Chloride  and  Picric  Acid  Solution  (Rabl). 

Saturated  aqueous  sol.  of  mercuric  chloride        1  part. 

Saturated  aqueous  sol.  of  picric  acid  1  part. 

Distilled  water         -  2  parts. 

This  is  excellent  for  hardening  mammalian  embryos. 
The  tissues  remain  in  the  fluid  8  to  12  hours,  are  then  trans- 
ferred to  70%  alcohol  until  the  picric  acid  is  washed  out 
of  the  tissue;  the  hardening  is  completed  in  95%  alcohol. 

Mueller's  Fluid   (Heinrich  Miiller). 

The  fluid  is  made  after  the  following  formula: 

Potassium  bichromate     .         .         .         .         2.5  parts. 

Sodium  sulphate      .  .         .  1  part. 

Water 100  parts. 

The  potassium  bichromate  and  sodium  sulphate  are 
to  be  ground  in  a  mortar.  They  dissolve  quickly  if  the 
water  is  heated.  A  large  quantity  of  the  fluid  may  be  kept 
on  hand  as  it  does  not  deteriorate  by  standing.  Miiller's 
fluid  is  especially  to  be  recommended  when  large  pieces  are 
to  be  hardened,  but  it  must  be  remembered  that  the  hard- 
ening takes  place  very  slowly.  Pieces  of  about  an  inch 
cube  harden  in  2  to  4  weeks,  of  two  inches  cube  1  to 
months,  and  larger  masses  proportionately  longer ;  a  human 
brain  for  instance  needs  to  be  in  the  fluid  from  6  to  8 
months.  Always  use  a  large  quantity  of  the  fluid,  and  change 
whenever  it  becomes  turbid.  Weigert  recommends  that  the 
jar  containing  the  tissue  to  be  hardened,  especially  if  it  be 
the  central  nervous  system,  be  kept  in  a  warm  oven  at  a 
temperature  of  30°  to  40°C. ;  a  spinal  cord  may  in  this  way  be 


—ISO- 
hardened  in  1  to  2  weeks.  After  hardening  in  Miiller's 
fluid  the  tissues  need  to  be  well  washed  in  flowing  water 
for  several  hours.  They  are  then  placed  into  75  %  alcohol 
for  2  or  3  days,  and  into  95  %  for  4  to  6  days ;  they  are  now 
ready  for  cutting.  Hans  Virchow*  recommends  that  the 
preparation  be  transferred  from  Miiller's  fluid  into  96% 
alcohol;  the  tissues  must,  however,  be  kept  in  the  dark. 

This  method  is  especially  useful  for  hardening  the 
central  nervous  system,  and  when  necessary  to  harden 
large  masses,  entire  organs,  tumors,  etc.  It  is  riot  used  to 
advantage  when  the  finer  structure  of  cells  is  desired. 

Nitric  Acid  (Benda). 

Benda  recommends  a  10%  solution  ;  in  this  the  tissues 
remain  from  24  to  48  hours,  are  then  transferred  to  Miiller's 
fluid  for  1  or  2  weeks,  thoroughly  washed  in  flowing  water 
for  several  hours ;  and  the  hardening  completed  in  graded 
alcohol.  This  method  gives  good  results  when  it  is  desired 
to  harden  an  entire  eye ;  the  HNO3  fixes  the  tissues,  and  it 
can  then  be  placed  into  Miiller's  fluid  without  collapsing. 

The  skin  and  scalp  are  also  well  hardened  after  this 
method. 

Picric  Acid. 

A  saturated  aqueous  solution  is  kept  on  hand.  Always 
filter  before  using.  The  tissue  needs  to  be  cut  into  small 
blocks ;  they  remain  in  the  fluid  for  1  to  3  days,  are  then 
rinsed  in  water,  and  placed  in  80%  alcohol,  which  must  be 
changed  as  often  as  it  becomes  yellow ;  as  soon  as  none  or 
very  little  of  the  acid  is  given  off,  place  in  95%  alcohol. 
Peripheral  nerves,  vessels,  elastic  cartilage,  and  fibro-carti- 
lage  are  well  hardened  in  this  way.  Fcetal  bones  are 
decalcified. 

Flemming's  Solution. 

One  of  the  best  of  hardening  solutions  is  a  mixture  of 
chromic,  osmic,  and  acetic  acid  in  the  following  propor- 
tions : 

*  Quoted    by  Rawitz  —  Leitfaden,    Histologjischer  Untersuch- 
ungen. 


—1 31— 

Osmic  acid  (2%  aqueous  sol.)  .         4  parts. 

Chromic  acid  (1%  aqueous  sol.)          .       15  parts. 

Glacial  acetic  acid        ....         1    part. 

The  solution  may  be  kept  on  hand  in  a  well-stoppered 
bottle.  Tissues  must  be  in  small  pieces,  one  dimension  of 
which  ought  not  to  be  more  than  one-twelfth  or  one-eighth 
of  an  inch.  The  chromic  acid  might  penetrate  larger  pieces, 
the  osmic  might  riot.  The  tissues  remain  in  the  solution 
about  24  hours,  are  then  thoroughly  washed  in  flowing 
water,  and  the  hardening  completed  in  graded  alcohol. 
This  solution  is  largely  used  in  hardening  tissues  for  cell 
division,  and  for  bringing  out  the  finer  details  in  the  struc- 
ture of  the  protoplasm  and  nucleus.  Unless  the  tissues 
are  well  washed  it  is  often  hard  to  stain  them. 
Hermann's  Solution. 

Hermann  uses  in  place  of  the  chromic  acid  in  Flem- 
ming's  solution  a  1  %  aqueous  solution  of  platinum  chloride, 
the  formula  reading  as  follows : 

Platinum  chloride  (1%  aqueous  sol.)         15  parts. 

Osmic  acid  (2%  aqueous  sol.)  .     2-4  parts. 

Glacial  acetic  acid        ....         1    part. 

The  pieces  need  to  be  small ;  they  remain  in  the  solu- 
tion for  24  to  48  hours,  are  then  washed  in  water,  and 
hardened  in  graded  alcohol.  Hermann's  fluid  is  used  with 
good  results  in  hardening  for  karyokinesis,  spermatogenesis, 
etc. 

Solution  of  Platinum  Chloride,  Osmic  Acid,  and  Mercuric 
Chloride. 

The  author  has  found  the  following  solution  a  very 
good  fixative : 

Platinum  chloride  (2%  aqueous  sol.)          3  parts. 

Osmic  acid  (2%  aqueous  sol.)  .         2  parts. 

Mercuric  chloride  (sat.  aqueous  sol.)  5  parts. 

It  is  especially  useful  for  hardening  the  testes  of  mam- 
malia. Very  small  pieces  are  to  be  hardened  for  about 
24  hours,  are  then  thoroughly  washed  in  flowing  water  for 
12  hours,  and  the  hardening  completed  in  graded  alcohol. 
In  testes  hardened  in  this  solution,  the  accessary  nucleus  is 
very  clearly  seen  in  nearly  every  cell ;  its  change  in  shape 
until  it  forms  the  "head  cap"  of  the  fully  developed 
spermatozoa  can  easily  be  traced. 


Methods  for  Decalcifying  Tissues. 


Kahlden*  gives  the  following  general  directions  to  be 
observed  when  decalcifying: — 

(1)  The  tissues  must  first  be  well  hardened  in  alcohol 
or  Muller's  fluid. 

(2)  A  large  quantity  of  the  decalcifying  fluid  needs  to 
be  used,  and  changed  frequently. 

(3)  After  decalcification  the  tissues  must  be  thoroughly 
washed  in  flowing  water  for  several  days. 

(4)  They  are  again  hardened  in  graded  alcohol,  after 
which  they  are  ready  for  cutting. 

Nitric  and  Hydrochloric  Acid. 

Use  the  following  proportions  : 

Nitric  acid  (10%  aqueous  solution)     .         1  part. 

Hydrochloric  acid  (1%  aqueous  solution)   1  part. 

The  decalcification  is  quite  rapid,  the  fluid  needs  to  be 
changed  every  second  or  third  day.  The  tissues  are  from 
time  4;o  time  taken  from  the  fluid  and  tested  by  pushing  a 
needle  into  the  bone,  and  if  it  enters  easily  and  without 
grating,  the  decalcification  may  ba  considered  complete. 
They  are  then  washed  in  flowing  water,  and  hardened  in 
graded  alcohol. 
Ebner's  Decalcifying  Fluid. 

The  following    formula  is  taken  from    Behren's  Ta- 
bellen : 

Sodium  chloride         ....         2.5  grms. 

Water 100.0  c.c. 

Alcohol 500.0  c.c. 

Hydrochloric  acid      ....         2.5  c.c. 

*Technik  der  Histologischen   Untersuchung  patolo^isch-ana- 
tomiscber  Prseparate.    Third  edition,  page  13. 


-133— 

This  solution  decalcifies  very  slowly,  but  without  in- 
jury to  the  tissues.  Large  quantities  (10  to  20  times  the 
bulk  of  the  tissue)  need  to  be  used,  and  one  or  two  c.c.  of 
hydrochloric  acid  are  daily  added  to  the  fluid  until  decal- 
cification  is  complete.  Wash  very  thoroughly  in  flowing 
water,  and  harden  in  graded  alcohol. 

Haug's  Chrom-Osmium  Solution. 

Osmic  acid  (1%  aqueous  solution)  .  10  c.c. 
Chromic  acid  (1%  aqueous  solution)  .  25  c.c. 
Water 60  c.c. 

Useful  for  decalcifying  the  cochlea ;  it  acts  very  slowly. 
Wash  in  flowing  water,  and  harden  in  alcohol. 

*Lepkowski's  Method. 

Small  pieces  of  tooth  or  bone  are  placed  in  the  follow- 
ing solution : 

Chloride  of  gold  (1%  aqueous  solution)  6  parts. 
Formic  acid  .....  3  parts. 

The  tissues  are  decalcified  in  two  or  three  days.  '  The 
chloride  of  gold  is  deposited  in  the  dentinal  tubules  or 
canaliculi,  so  that  the  tissue  will  be  both  stained  and 
decalcified  at  the  same  time. 

*Anatomischer  Auzeiger,  1892. 


Impregnation  of  Tissues. 


Silver  Nitrate. 

This  is  used  for  staining  the  endothelial  membranes 
lining  blood-vessels  and  serous  cavities.  An  albumenate  of 
silver  is  formed  with  the  intercellular  cement  between  the 
endothelial  cells;  the  silver  is  reduced  on  exposing  to  sun- 
light. A  1%  aqueous  solution  of  the  silver  nitrate  is 
used.  In  making  a  preparation  of  an  endothelial  mem- 
brane, the  peritoneum  of  a  frog  may  be  selected.  Make  a 
small  opening  through  the  abdominal  wall  of  a  frog  near 
the  sternum,  inject  10  to  20  c.c.  of  the  1%  solution  of 
silver  nitrate ;  while  injecting,  the  abdomen  is  gently 
kneaded,  so  that  the  fluid  may  be  well  distributed  in  the 
abdominal  cavity.  In  15  to  20  minutes  the  abdomen  is 
opened,  the  intestinal  canal  with  the  mesentery  is  removed, 
the  latter  (without  removing  from  the  intestines)  is  pinned 
out  on  a  cork  board,  is  now  immersed,  preparation  side  up, 
in  80%  alcohol  (it  will  be  necessary  to  fix  small  lead 
weights  to  the  cork  to  bring  it  under  the  alcohol),  and 
placed  in  the  sunlight ;  as  soon  as  the  tissue  shows  a  brown 
color  it  is  ready  for  study.  It  may  be  mounted  in  gum 
glycerine  or  balsam. 

Another  method  is  to  carefully  remove  the  peritoneum, 
pleura,  or  pericardial  sack,  and  spread  out  on  a  clean,  dry 
slide,  to  which  the  tissue  will  adhere  quite  firmly  if  set  aside 
for  a  few  moments.  The  slide  with  the  tissue  on  it  may 
then  be  placed  in  the  silver  nitrate  solution  and  treated 
as  above.  Connective  tissue,  .when  stained  in  silver  nitrate 
to  show  the  cell  spaces,  may  be  prepared  in  the  same  way. 
If  it  is  desired  to  mount  the  tissue  in  balsam,  it  may, 


—135— 

after  the  reduction  of  the  silver  has  taken  place,  be 
dehydrated  in  absolute  alcohol,  cleared  in  oil  of  bergamot, 
and  mounted  in  balsam. 

SILVER    NITRATE   AND     BICHROMATE    OF    POTASH 
METHOD  (Golgi,  Ramon  y  Cajal,  andLenhossek). 

This  is  a  method  used  for  staining  nerve  cells  and  their 
processes  both  in  the  central  and  peripheral  nervous  system. 
The  formulae  here  given  are  taken  from  Lenhossek's  Referat 
in  Fortschritte  der  Medicin,  August  and  September,  1892. 

Golgi's  Slow  Method. 

Small  pieces  of  brain  or  spinal  cord  are  hardened  in  a 
2%  solution  of  bichromate  of  potassium  from  20  to  30 
days;  are  then,  without  washing,  transferred  to  a  -J-%  solu- 
tion of  silver  nitrate,  in  which  they  remain  from  24  to  48 
hours;  or  in  place  of  the  silver,  a  5%  solution  of  bichloride 
of  mercury  may  be  used ;  in  this  they  remain  from  2  to  4 
weeks. 

The  Mixed  Golgi  Method. 

The  tissues  to  be  hardened  and  stained  are  placed  in  a 
large  quantity  (20  to  30  times  the  bulk  of  the  tissues  used) 
of  the  following  solution  : 

Bichromate  of  potassium  .  (1%  aqueous  sol.)  8  parts. 
Osmic  acid  .  ..  .  (1%  aqueous  sol.)  1  part. 

In  this  they  remain  four  or  five  days ;  are  then  trans- 
ferred to  0.75%  solution  of  silver  nitrate  for  24  to  30  hours. 

Rapid  Method  (Ramon  y  Cajal). 

The  following  solution  is  now  largely  used : 

Bichromate  of  potassium  (3  to  5%  sol.)          4  parts. 
Osmic  acid          .         .        (1%  aqueous  sol.)    1  part. 

If  it  is  desired  to  stain  neuroglia  cells,  allow  the  tis- 
sues to  remain  in  the.  solution  2  or  3  days ;  if  nerve  cells, 
3  to  5  days ;  if  nerve  fibres  and  collateral  branches,  5  to  7 
days.  They  are  then  transferred  to  a  1%  aqueous  solution 
of  silver  nitrate,  in  which  they  remain  from  24  to  36  hours. 
Lenhossek  adds  one  drop  of  formic  acid  to  200  c.  c.  of  the 
silver  solution. 


These  methods  give  the  best  results  when  embryonic 
tissues,  or  tissues  taken  from  newly  born  or  young  animals, 
are  employed. 

Often  when  the  methods  above  noted  do  not  give  good 
results  at  the  first  trial,  they  may  with  success  be  repeated 
on  the  same  tissue.  If  after  the  tissues  have  been  2  or  3 
days  in  the  silver  nitrate,  the  trial  sections  (free-hand 
sections  cut  from  the  blocks,  mounted  and  examined  in 
95%  alcohol)  show  no  staining,  they  may  again  be 
placed  in  the  bichromate  of  potassium  solution  for  several 
days,  and  then  again  into  silver.  Good  results  follow  a 
second  and  even  the  third  trial. 

Sections  are  cut  into  95  %  alcohol ;  they  may  be  made 
either  free-hand  or,  after  surrounding  with  paraffin  or 
embedding  in  celloidin,  cut  with  the  microtome.  From  the 
alcohol,  sections  are  placed  for  15  minutes  into  creosote 
then  washed  from  3  to  5  minutes  in  oil  of  turpentine, 
out  of  which  they  are  taken  and  arranged  on  the  slide. 
The  excess  of  oil  of  turpentine  is  removed  with  filter  paper, 
and  the  sections  are  covered  with  balsam.  The  slide  is  now 
carefully  heated  over  an  alcohol  flame,  until  the  balsam  be- 
comes so  thick  that  on  cooling,  it  at  once  hardens  (3  to  5 
minutes  of  careful  heating  are  required,  allow  no  bubbles 
to  form) ;  while  the  balsam  is  yet  warm  cover  with  a  cover 
glass  which  has  been  passed  through  the  flame  several 
times ;  after  cooling,  the  preparation  is  ready  for  study. 
Mounted  in  this  way,  Golgi  preparations  do  not  fade.* 

Gold  Chloride  (Ranvier's  Lemon  Juice  Method). 

This  method  is  especially  useful  for  staining  the  nerve- 
ending  involuntary  muscle. 

Place  small  pieces  of  a  short  muscle  (intercostal  of  a 
snake  or  guinea  pig)  in  filtered  lemon  juice  for  10  to  15 
minutes  (or  until  the  tissue  becomes  transparent),  wash 
hastily  in  distilled  water  and  transfer  the  tissue  to  a  1% 
solution  of  chloride  of  gold,  in  which  they  remain  for  15  to 
20  minutes.  The  tissue  is  then  again  hastily  washed  in 

*  Huber.  Aiiatomischer  Anzeiger.  VII  Jahrgang  (1892), 
No.  18. 


-137- 

distilled  water,  and  placed  in  a  20%  solution  of  formic  acid, 
in  which  they  remain  in  the  dark  for  24  to  48  hours. 
Tease  a  small  portion  of  the  muscle  in  glycerine,  and  if  the 
motor  endings  are  stained,  transfer  the  tissue  to  glycerine. 
Tease  and  mount  in  glycerine. 


Methods  for  Injecting. 


When  it  is  desired  to  bring  out  prominently  the  rela- 
tion between  the  blood  vessels  and  the  other  elements  of  a 
tissue  or  an  organ,  it  is  necessary  to  inject  the  vessel. 
This  is  best  done  by  means  of  substances  which  are  fluid 
when  warm,  but  harden  on  cooling.  Gelatin,  which  has 
been  colored  with  some  dye,  is  usually  employed.  The 
directions  for  making  two  such  injecting  masses  are  here 
given.  Much  experience  is  required  to  inject  successfully ; 
too  much  space  would  be  required  to  go  fully  into  the 
methods  used.  They  are  demonstrated  in  the  course  on 
methods. 

Carmine  Gelatin  (Gerlach). 

The  formula  is  taken  from  Behren's  Tabellen. 

Carmine  .         .         ....         10  grms.) 

Ammonium  hydrate         .         .         .         .  1  c.  c.     >    I. 

Water  .         .         .         .         .         .  8  c.  c.    ) 

Gelatin  .         .         .         .         .         .         12  grmsJ    TT 

Water  16  c.  c.    | 

The  gelatin  is  -cut  into  fine  pieces,  placed  in  an  evapo- 
rating dish,  and  the  water  added;  in  this  the  pieces  soak 
about  12  hours.  The  gelatin  is  then  dissolved  over  a 
water-bath.  To  solution  II,  add  solution  I  (which  is  pre- 
pared by  dissolving  the  carmine  in  the  ammonium  hydrate 
and  water  over  a  water-bath),  slowly,  while  constantly  stir- 
ring. The  mass  is  now  alkaline,  and  unless  neutralized 
would  stain  the  bloodvessels  and  surrounding  tissue.  The 
neutralization  is  accomplished  by  means  of  glacial  acetic 
acid,  which  is  added  drop  by  drop  until  no  ammonia  is  de- 
tected by  the  sense  of  smell,  stirring  well  after  every  drop. 


—139— 

It  will  also  be  noticed  that  the  mass  changes  its  color,  be- 
coming a  brighter  red.  If  the  mass  becomes  too  acid  it 
appears  granular  if  a  drop  is  examined  under  the  micro- 
scope ;  in  this  state  it  is  not  entirely  useless,  but  the  result- 
ing injection  is  never  so  good  as  when  the  mass  is  neutral. 
Before  using,  the  mass  must  be  strained  through  a 
piece  of  flannel  which  has  been  dipped  in  hot  water.  The 
canula,  syringe,  and  animal  must  be  kept  warm  during  the 
injection.  If  the  entire  animal  is  to  be  injected,  the  canula 
is  to  be  tied  into  the  arch  of  the  aorta,  through  the  left 
ventricle  ;  if  a  single  organ,  the  canula  is  tied  into  its  main 
artery.  As  soon  as  the  injection  mass  appears  in  the 
accompanying  vein,  this  vein  is  firmly  tied.  After  the 
tissues  have  been  fully  injected,  the  animal  or  organ  is  set 
aside  in  a  cold  place  until  the  injection  congeals,  the  tissues 
are  then  removed,  cut  into  small  pieces,  and  hardened  in 
alcohol.  These  may,  before  embedding,  be  stained  in 
Delafield's  hsematoxylin. 

Berlin  Blue  Mass  (Harting,  as  given  by  Rawitz) . 

One  part  of  oxalic  acid  is  "  rubbed  up  "  in  a  glass 
mortar;  to  this  is  added  one  part  of  Berlin  blue,  and,  while 
constantly  stirring,  12  parts  of  water.  To  an  equal  quantity 
of  warm  gelatin  solution  (made  as  above  directed)  add  the 
Berlin  blue  solution  slowly,  continually  stirring.  Filter 
through  a  piece  of  flannel  before  using. 

Inject  while  the  solution  is  warm.  After  the  tissues 
are  cold,  harden  in  alcohol.  The  tissues  may  be  stained  in 
alum  or  borax  carmine. 


Methods  for  Embedding  and  Cutting 
Sections. 


Free  Hand  Cutting. 

It  is  usually  necessary  to  surround  the  tissues  to 
be  cut  with  some  material,  which  gives  no  resistance  to, 
and  does  not  injure  the  knife,  at  the  same  time  is  firm 
enough  to  give  support.  Pathologists  have  for  a  long 
time  used  small  strips  of  amyloid  liver  which  had  been 
hardened  in  alcohol  or  Muller's  fluid.  If  these  can  not 
be  obtained,  pig's  or  calf's  liver  will  answer.  Elder- 
pith  is  also  used.  The  little  blocks  of  liver  tissue,  or  the 
elder-pith  rods,  are  divided  into  two  parts,  between  which 
the  tissue  to  be  cut  is  placed.  It  can  now  be  firmly 
held  between  the  thumb  and  the  index  finger  of  the  left 
hand.  A  razor,  which  is  flat  on  one  side  (the  under  while 
in  use),  is  employed.  Its  upper  surface  is  well  covered 
with  80%  alcohol.  Try  to  make  the  section  with  one  con- 
tinuous cut,  resting  the  blade  of  the  knife  on  the  index 
finger.  Sections  which  answer  for  purposes  of  orientation 
can  easily  be  made  in  this  way.  They  are,  however,  not  to 
be  compared  with  the  ones  that  can  be  cut  with  the 
microtome  when  the  tissues  are  properly  embedded. 

Embedding  Tissues. 

A  great  many  methods  for  embedding  tissues  are  in 
use,  the  principle  involved  is  the  same  in  all.  In  the  one 
case  the  tissues  are  permeated  with  substances  that  are 
fluid  when  warm  and  become  hard  enough  to  cut  on  cool- 
ing; in  others  again,  the  embedding  mass  hardens,  on  the 
evaporation  of  a  solvent  which  was  used  to  bring  them  to  a 
fluid  state.  The  methods  for  embedding  in  celloidin,  in 


paraffin,  and  in  a  solution  of  gum  arable  (for  the  freezing 
microtome)  are  hfjre  given.  It  is  deemed  beyond  the  scope 
of  these  notes  to  go  into  the  methods  for  cutting  sections; 
they  will  be  taught  in  the  laboratory. 

Embedding  for  Cutting  with  the  Freezing  Microtome. 

To  prepare  the  tissues  for  cutting  on  the  freezing 
microtome,  remove  the  alcohol  from  the  hardened  tissues 
by  allowing  them  to  remain  in  water  about  eight  hours, 
then  transfer  to  a  solution  of  gum  arabic  (the  solution 
should  be  about  as  thick  as  syrup).  It  is  made  by  dissolv- 
ing the  gum  arabic  in  hot  water,  and  straining  through  a 
cloth;  cool  before  using.  In  about  six  hours  they  are 
permeated  with  the  gum  arabic  and  are  ready  for  freezing. 
Sections  need  to  be  cut  into  warm  distilled  water,  which 
removes  the  gum  arabic.  Before  staining  or  placing  in 
alcohol  they  must  again  be  washed  in  distilled  water. 

Celloidin  or  Collodium  Embedding.* 

A  stock  solution  of  celloidin  or  collodium  is  kept  on 
hand.  This  is  made  by  adding  celloidin  or  collodium  to  a 
mixture  of  equal  parts  of  absolute  alcohol  and  ether  until 
a  thick  solution  is  obtained.  The  stock  solution  needs  to 
be  kept  in  a  well-stoppered  bottle,  as  it  thickens  on  the 
evaporation  of  the  alcohol  and  ether.  The  steps  for  em- 
bedding tissues  are  the  following  : — 

(1)  From  the  95%  alcohol  the  tissues  are  placed  into 
absolute  for  24  to  48  hours. 

(2)  Into  a  mixture  of  equal  parts  of.  absolute  alcohol 
and  ether  from  1  to  2  days. 

(3)  Into  a  thin  solution  of  celloidin,  consisting  of  one 
part  of  the    stock  solution  and   two  parts  of    the  alcohol 
and  ether  mixture    (equal   parts) ;    in  this  they   remain, 
according  to  the  size  of  the  pieces  embedded,  from  2  or  3 
days  to  as  many  weeks. 

(4)  Into  the    stock  solution    for   an  equal   length  of 
time.     The  tissues  are  ready  for  further  treatment  as  soon 

*  The  methods  for  embedding  in  celloidin  and  collodium  are 
the  same,  and  the  results  seem  to  the  author  equally  good ;  collo- 
dium is  cheaper.  The  steps  here  given  for  the  one  will  answer  for 
the  other. 


—  142— 

as  they  are  thoroughly  permeated  with  the  celloidin  ;  no 
definite  time  can  be  fixed  for  this.  Loose  tissues,  such  as 
lung,  are  permeated  in  3  or  4  days;  skin  in  2  or  3  weeks; 
brain  or  spinal  cord,  especially  if  the  pieces  are  large,  in  3 
to  6  weeks. 

As  soon  as 'permeation' seems  complete,  some  of  the 
stock  solution  is  poured  into  a  flat  glass  dish  (enough 
to  cover  well  the  tissues  to  be  embedded),  into  this  the  tis- 
sues are  brought,  and  if  several  pieces  are  to  be  embedded, 
they  are  to  be  so  arranged  that  a  small  area  of  celloidin 
(one-fourth  of  an  inch  wide)  surrounds  each  piece.  The 
dish  is  now  covered  with  a  glass  plate,  and  placed  under  a 
bell-jar.  The  alcohol  and  ether  evaporate  very  slowly,  as 
this  goes  on  the  celloidin  hardens.  In  case  the  glass  plate 
fits  tightly,  push  it  to  one  side,  thus  leaving  a  small  space 
uncovered.  Several  days  are  required  for  the  celloidin  to 
become  hard.  As  soon  as  the  mass  is  so  firm  that  it  can 
not  be  indented  with  the  finger,  it  is  removed  from  the 
glass  dish.  It  can  now  be  cut  into  a  square  block  contain- 
ing the  tissue;  or  into  several,  if  a  number  of  pieces  were 
embedded.  These  celloidin  blocks  are  to  be  fixed  to  small 
cylinders  of  hard  wood.  The  diameter  of  the  wooden 
blocks  should  be  a  little  larger  than  the  celloidin  block, 
and  about  one-half  an  inch  long.  This  is  done  by  immers- 
ing one  end  of  the  wooden  cylinder  in  the  stock  solution ; 
a  layer  of  celloidin  is  in  this  way  spread  over  one  end  of 
the  cylinder  of  wood.  The  celloidin  block  is,  after  having 
been  immersed  in  equal  parts  of  alcohol  and  ether  for  about 
5  minutes,  pressed  against  the  layer  of  celloidin  on  the 
wooden  block,  and  then  placed  under  a  bell-jar  for  about 
an  hour.  It  is  then  transferred  to  70%  alcohol  for  several 
hours,  at  the  end  of  which  time  it  is  ready  for  cutting. 

When  cutting  sections  of  a  tissue  embedded  in  celloidin, 
the  knife  should  be  moistened  with  80%  alcohol;  sections 
are  cut  into  weak  alcohol  or  distilled  water.  The  celloidin 
blocks,  even  after  they  are  fixed  to  the  wooden  cylinders, 
may  be  kept  for  a  long  time  in  70%  alcohol. 

The  celloidin  method  is  now  very  largely  used ;  it  is 
especially  useful  for  cutting  sections  of  the  central  nerv- 


—143— 

ous  system,  of  an  entire  eye,  or  tissues  or  organs  con- 
taining much  fibrous  tissue.  It  may  be  used  whenever  not 
very  thin  sections  (less  than  10,"-.  to  15;;-.)  are  required. 
The  celloidin  or  collodium  need  not  be  removed  before 
staining  or  mounting ;  and  unless  the  aniline  dyes  are  used 
for  coloring,  very  little  stain  is  taken  by  the  celloidin.  The 
sections  need  to  be  cleared  in  oil  of  bergamot,  as  the  oil  of 
cloves  dissolves  the  celloidin.  The  following  method  for 
staining  and  mounting  celloidin  sections  in  series,  is  recom- 
mended by  Weigert : — * 

(1)  A  clean  glass  plate  is  covered  with  a  thin  layer  of 
a  solution  of  collodium ;  this  is  to  be  spread  out  as  evenly 
as    possible  over  the  entire    surface.      The  plate   is    now 
placed  on  edge,  and   the  collodium   allowed  to   dry,  care 
being  taken  to  keep  the  dust  from  it. 

(2)  A  section  is  placed  on  a  strip  of  "closet  paper," 
near  one  end.  .  Succeeding  sections,  as   soon  as  cut,  are 
placed   to  the  right  of  it.      They  are  removed  from  the 
knife  to  the  strip  of  paper,  by  holding  the  paper  extended 
under  the  knife,  and  slipping  the  section  onto  it.     To  keep 
the  sections  from  drying,  the  strip  of  paper  is,  after  the 
removal  of  every  section,  placed  in  a  flat  dish,  in  which 
several  layers  of  filter  paper,  have   been    spread  out  and 
thoroughly  saturated  with  80%  alcohol.     On  each  strip  of 
paper  is  arranged  only  one  row  of  sections ;  the  strips  are 
kept  in  the  dish  in  the  order  used. 

(3)  As  soon  as   a  number  of  strips  have  been  covered 
with   sections,  they   are   arranged,   sections  downward,  on 
the  layer  of  collodium  above  described,  and  gently  pressed 
to  it.     The  strips  of  the  papei   can  now  be  removed,  the 
sections  adhering  to  the  layer  of  collodium.     Several  layers 
of  filter  paper  are  now  pressed  over  the  sections,  in  this 
way  removing  as  much  of  the  alcohol  as  possible. 

(4)  Before  the  sections  have  time  to  dry  a  layer  of 
the  collodium  solution  is  poured  over  them,  equally  dis- 
tributed, and  allowed  to  dry.     As   soon  as  dry  the  plate 
may  be  placed  into  80%  alcohol,  where  it  may  be  kept,  or 

*  Taken  from  Rawitz  Leitfaden  der  histologischen  Uutersuch- 
ungen.    Page  37. 


144" 

into  the  stain;  the  layer  of  collodium  containing  the  sec- 
tions then  separates  from  the  glass  plate,  and  it  can  be 
treated  as  a  single  section. 

Paraffin  Embedding. 

Undoubtedly  the  paraffin  method  of  embedding  is  of 
all  methods  the  most  satisfactory  for  general  work  in  his- 
tology, embryology,  and  pathology.  Only  very  few  tissues 
met  with,  can  not  to  advantage  be  embedded  in  this  way. 
Exceedingly  thin  sections  can  be  cut  when  the  tissues  are 
properly  embedded  in  paraffin,  and  as  the  paraffin  is 
always  removed  from  sections  before  staining,  it  has  none 
of  the  disadvantages  of  the  celloidin  method.  The  tissues 
after  thorough  dehydration  are  placed  for  a  longer  or 
shorter  time  into  a  fluid  which  mixes  readily  with  alcohol 
on  the  one  hand  and  is  a  good  solvent  for  paraffin  on  the 
other  hand.  The  method  used  in  this  laboratory  is 
briefly  as  follows  : —  • 

(1)  The  tissues  are  thoroughly  dehydrated  in  absolute 
alcohol.     This  takes  from  2  to  24  hours,  depending  on  the 
size  of  the  pieces,  the  density  of  the  tissue  and  the  exteixt 
of  dehydration  before  placing  in  absolute  alcohol. 

(2)  The  tissues  are  then  transferred  to  toluol  or  xylol 
(chloroform,  turpentine,  or  oil  of  origanum  may  be  used) 
for  4  to  24  hours.     Toluol  is  preferred  as  it  mixes  very 
readily  with  absolute  alcohol  and  is  one  of  the  very  best 
solvents  of  paraffin.     In  case  the  tissues  are  not  thoroughly 
dehydrated    before    placing    in  the  toluol  or  xylol,  these 
fluids  become  milky  and  the  tissue  should  then  be  again 
transferred  to  absolute   alcohol   until  the   dehydration   is 
complete. 

(3)  The  tissues  are  then  placed  into  melted  soft  paraf- 
fin in  which  they  remain  for  1  to  12  hours,  again  depend- 
ing on  the  size  of  the  piece  of  tissue  to  be  embedded,  its 
density,  etc.     Soft  paraffin  has  a  melting  point  of  about  38° 
to  42°  C.     The  cup,  jar,  or  bottle  containing  this  paraffin 
is  kept  in  a  warm  oven,  the  temperature  of  which  can  be 
regulated;  it  should  not  exceed  50°  to  52°  C.     if  delicate 
tissues,  embryos,  etc.,  are  to  be  embedded,  it  is  well  to  add 
from  time  to  time  a  few  small   pieces  of  unmelted  soft 


—145— 

paraffin  to  the  toluol  or  xylol  containing  the  tissue  to  be 
embedded ;  the  dish  or  bottle  containing  said  tissue  can  at 
this  time  be  placed  in  the  warm  oven.  In  this  way  the 
change  from  the  toluol  or  xylol  to  the  melted  soft  paraffin 
can  be  made  very  gradual,  and  there  will  be  less  danger  of 
causing  any  shrinkage  in  the  tissue. 

(4)  From  the  soft  paraffin,  the  tissues  are  transferred 
to  melted  hard  paraffin,  which  is  usually  a  mixture  of  equal 
parts  of  soft  paraffin  and  a  paraffin  with  a  melting  point  of 
about  52°  to  55°  C.,  and  answers  very  well  for  ordinary 
room  or  laboratory  temperature.  During  the  summer 
months,  it  may  be  necessary  to  use  two  parts  of  the  paraf- 
fin with  55°  C.  melting  point,  and  only  one  of  the  soft,  and 
in  very  warm  weather  even  less  of  the  soft. 

In  very  warm  weather  the  author  has  often  used  the 
following  mixture  with  success  : 

Soft  paraffin  ...         1  part  by  weight. 

Hard  paraffin  4  parts  "       " 

Spermaceti  ...         1  part    "       " 

In  the  hard  paraffin  the  tissues  remain  from  2  to  24 
hours,  of  course  in  the  warm  oven.  It  is  essential  that  the 
melted  paraffins  while  in  use  for  embedding  should  be  at 


Fig.  I.  Simple  paraffin  bath;  A,  copper  plate;  B,  paraffin  tray;  C, 
loop  of  filter  paper;  D,  area  of  melted  paraffin;  E,  area  of  un  melted  paraffin; 
F,  tripod;  G,  flame. 

a  constant  temperature ;  this  can  easily  be  done  when  a 
water-bath  with  temperature  regulator,  such  as  is  found  in 
most  laboratories,  is  at  hand.  A  very  simple  apparatus, 

and  one  that  meets  the  requirements  quite  well,  is  shown 
10 


— 146 — 

in  Fig.  I.  It  consists  of  a  tripod  (F) ;  a  copper  plate  (A) 
about  fifteen  inches  long,  -five  inches  wide,  and  one-eighth 
to  three-sixteenths  of  an  inch  thick ;  and  two  tin  trays 
(B)  (only  one  is  shown  in  the  diagram);  these  are  ten 
inches  long,  two  wide,  and  three  deep.  They  are  partly 
filled  with  paraffin ;  the  one  with  soft,  the  other  with  the 
hard.  If  an  alcohol  lamp  or  a  Bunsen  burner  be  so  placed 
that  the  end  of  the  flame  (G)  touches  one  end  of  the  cop- 
per plate,  as  shown  in  diagram,  and  the  trays  containing 
the  hard  and  soft  paraffin  be  placed  on  the  copper  plate 
toward  the  end  away  from  the  flame,  it  will  be  found  that 
after  a  short  time,  fifteen  to  thirty  minutes,  the  paraffin  in 
the  end  of  the  trays  near  the  flame  will  be  melted  while  in 
the  other  end  it  is  yet  hard,  by  reason  of  the  fact  that  the 
flame  end  of  the  copper  plate  has  a  higher  temperature 
than  the  opposite  end.  With  a  little  patience  the  trays  may 
be  so  adjusted,  by  moving  them  toward  or  away  from  the 
flame,  that  about  half  of  the  paraffin  will  be  melted,  the 
rest  not.  As  shown  in  the  diagram,  D  represents  the  area  of 
melted  paraffin,  E  of  the  unmelted.  It  of  course  stands 
to  reason,  that  if  the  two  trays  be  placed  side  by  side  on 
the  copper  plate,  the  one  containing  the  hard  paraffin  needs 
to  be  nearer  the  flame  than  the  one  containing  the  soft,  to 
obtain  in  each  an  area  of  melted  and  unmelted  paraffin. 
In  either  tray,  the  area  of  unmelted  paraffin  acts  as  a  ther- 
mometer, the  adjoining  melted  paraffin  must  have  a  tem- 
perature, which,  when  expressed  in  degrees,  is  about  the 
melting  point  of  the  paraffin  in  question;  about  40°  C.  for 
the  soft,  50°  C.  for  the  hard.  It  is  not  advisable  to  allow 
the  tissues  to  rest  on  the  bottom  of  the  tray.  A  loop  can 
easily  be  made  with  a  strip  of  filter  paper  about  two  inches 
wide ;  this  is  supported  from  a  wire  or  glass  rod,  and  allowed 
to  hang  in  the  area  of  melted,  near  the  edge  of  the 
unmelted  paraffin,  C  in  the  figure.  The  tissues  are  placed 
on  the  filter  paper. 

When  the  tissues  are  thoroughly  permeated  with  the 
hard  paraffin,  a  rectangular  trough  is  made  with  two  metalic 
L's,  resting  on  a  glass  plate,  and  filled  with  melted  hard 
paraffin.  Into  this  the  tissue  is  placed  by  means  of  a  pair 
of  small  forceps,  which  before  using  were  warmed  in  a 


—  147— 

flame.  The  piece  of  tissue  is  placed  in  one  end,  and  so 
arranged  that  the  plane  in  which  it  is  to  be  cut,  is  at  right 
angles  to  the  long  axis  of  the  trough.  The  paraffin  is  now 
allowed  to  cool,  and  as  soon  as  a  film  forms  over  it,  the 
trough  is  placed  in  cold  water;  this  quickly  congeals  the 
paraffin.  The  metalic  L's  are  removed ;  the  paraffin  block 
can  now  be  taken  from  the  glass  plate,  and  is  ready  for  cut- 
ting. The  knife  used  for  cutting  paraffin  sections  must  be 
dry,  and  if  perfectly  embedded  very  thin  sections  can  be 
cut,  thinner  than  when  embedded,  after  any  other  method. 

Before  mounting  or  staining  paraffin  sections  it  is 
always  necessary  to  remove  the  paraffin.  Before  doing 
this  it  is  advisable  to  fix  the  section  to  the  slide  or  cover 
glass  by  means  of  one  of  the  many  methods  now  in  use. 
Sections  stuck  to  slide  or  cover  can  then  be  stained  with- 
out in  the  least  destroying  the  relation  of  the  elements  of 
the  tissue.  The  contents  of  tubules,  of  small  cysts  or 
spaces  do  not  fall  out  during  the  process  of  staining  and 
mounting. 

Of  the  many  methods  used  for  fixing  sections  to  slide 
or  cover  glass,  the  following  have  been  found  by  the  author 
to  be  most  reliable.  It  must  be  remembered  that  sections 
will  not  stick  well  unless  the  slide  or  cover  glass  is 
thoroughly  clean.  In  this  laboratory  they  are  cleaned  by 
placing  the  slides  or  cover  glasses  in  strong  sulphuric  acid 
for  10  to  15  minutes,  then  wash  thoroughly  in  flowing 
water,  next  place  them  for  a  few  minutes  in  glacial  acetic 
acid,  again  wash  in  flowing  water  until  all  the  acid  has 
been  removed,  pour  off  the  water,  and  add  95%  alcohol, 
from  which  the  slides  or  covers  are  wiped  with  a  clean  piece 
of  old  linen. 

Albumen  Fixative  (Mayer). 

The  albumen  fixative  consists  of  equal  parts  of  white 
of  egg  and  glycerine.  It  is  prepared  by  chopping  the  white 
of  an  egg  with  a  pair  of  scissors,  then  straining  it  through 
muslin  or  linen;  it  is  now  mixed  with  an  equal  quantity 
of  glycerine.  The  glycerine  and  white  of  egg  are  to  be 
thoroughly  mixed  by  stirring  with  a  glass  rod,  and  filtered. 


— 148— 

A  small  drop  of  the  albumen  fixative  is  placed  on  a 
slide,  and  spread  in  a  very  thin  layer  with  a  clean  glass  rod, 
or  better,  with  a  dry  and  clean  finger.  The  section  is  now 
placed  on  the  albumen  fixative,  and  pressed  to  the  slide. 
If  the  section  is  stained,  the  paraffin  is  melted  by  holding 
the  slide  or  cover  glass  over  an  alcohol  flame,  or  by  plac- 
ing them  on  a  water  bath,  until  the  paraffin  begins  to  melt; 
then  cover  with  a  few  drops  of  oil  of  turpentine,  which  dis- 
solves the  paraffin ;  remove  excess  of  oil  with  filter  paper, 
and  mount  in  balsam.  If  it  is  desired  to  stain  the  sections 
after  they  are  fixed  to  the  slide  or  cover  glass,  the  follow- 
ing steps  are  taken  to  remove  the  paraffin  : — 

1.  Heat  quickly  over  the  flame  until  the  paraffin  be- 
gins to  melt. 

2.  Place  the  slide  or  cover  glass  into  toluol  or  xylol 
until  the  paraffin  is  dissolved. 

3.  Transfer  to  absolute  alcohol  for  3  to  5  minutes; 
this  removes  the  toluol  or  xylol. 

4.  Transfer  to  95%  alcohol  for  5  minutes. 

5.  Transfer  to  70%  alcohol  for  several  minutes. 

6.  Remove  alcohol  by  placing  slide  or  cover  into  dis- 
tilled water.     Sections  can  now  be  stained. 

It  is  often  quite  difficult  to  remove  folds  from  paraffin 
sections  (especially  if  sections  are  large)  before  fixing 
to  the  slide  or  cover  glass  with  albumen  fixative;  the 
author  has  used  with  success  this  simple  method  ; 

An  evaporating  dish  is  partly  filled  with  distilled 
water;  on  this  the  sections  are  placed.  The  water  is  now 
slowly  heated  by  holding  the  evaporating  dish  over  an 
alcohol  flame.  It  will  be  noticed  that  as  the  temperature 
of  the  water  is  raised,  the  paraffin  in  and  about  the  sec- 
tions begins  to  soften,  and  the  sections  spread  out  over  the 
surface  of  the  water.  The  water  is  heated  until  all  folds 
are  obliterated.  Care  should  be  taken  not  to  get  the  water 
hot  enough  to  melt  the  paraffin.  A  slide  or  cover  glass,  on 
which  a  thin  layer  of  the  albumen  fixative  is  spread,  is 
now  passed  under  one  of  the  sections;  on  it  the  section  is 
caught  and  withdrawn  from  the  water.  Allow  all  the 
water  to  evaporate,  this  usually  takes  8  to  12  hours,  at  the 


end  of  which  time  the  paraffin  may  be  removed  as  above 
described.  This  method  is  very  useful  for  mounting  serial 
sections. 

Gaule's  50%   Alcohol  Method. 

A  few  drops  of  50%  alcohol  are  placed  on  a  slide  or 
cover  glass;  on  this  the  sections  are  placed.  As  the  alcohol 
evaporates,  the  sections  are  fixed  to  the  slide  or  cover; 
12  to  24  hours  are  required,  and  it  is  best  to  place  them  in 
a  warm  oven  at  a  temperature  of  40D  C.  The  paraffin  is 
removed  in  the  same  manner  as  when  sections  are  fixed 
with  albumen  fixative.  The  slide  or  cover  must  be  very 
clean,  even  then  the  sections  are  often  loosened  while  the 
paraffin  is  being  removed.  Sections  from  alcohol  or  subli- 
mate hardened  tissues  seem  to  be  most  firmly  fixed. 

Dextrin  Method  of  Fixing  Sections. 

This  method  is  to  be  recommended  for  class  purposes, 
where  30  to  50  or  even  more  preparations  have  to  be  stained 
in  section. 

The  following  two  solutions  are  kept  on  hand : — 
Sol.  No.  1. 

A  solution  of  equal  parts  of  white  sugar  and 

boiling  distilled  water  .  .  300  c.  c. 

A  solution  made  with  equal  parts  of  dextrin 

and  distilled  water  .  .  100  c.  c. 

Absolute  alcohol  .  .  .  200  c.  c. 

Mix  the  sugar  and  dextrin  solutions  in  a  mortar,  and 
add  very  slowly,  while  constantly  stirring,  the  absolute 
alcohol.  Filter  through  fine  muslin,  and  keep  in  a  wide- 
mouthed  bottle.  Place  in  the  cork  a  broad  camel's-hair 
brush. 

Sol.  No.  2. 

Photoxolyn  (dry)  or  good  celloidin  or  gun 

cotton     .....  10  grms. 

Absolute  alcohol  .         .         .         .         .  .  100  c.  c. 

Ether 500  c.  c. 

The  sections  to  be  stained  are  cut  in  paraffin,  and 
arranged  on  a  piece  of  paper.  A  clean  glass  plate  is  coated 


—ISO— 

with  a  thin  layer  of  the  dextrin  solution.  The  paraffin 
sections  are  then  arranged  in  rows  on  the  layer  of  dextrin, 
and  pressed  against  the  plate  with  the  finger.  The  plate 
with  the  sections  fixed  to  it  is  now  placed  in  the  warm  oven, 
the  temperature  of  which  should  not  exceed  40°  C.,  where 
it  remains  from  4  to  24  hours.  The  plate  is  then  taken 
from  the  warm  oven,  warmed  over  a  flame  until  the  paraf- 
fin about  the  sections  begins  to  melt,  and  transferred  to  a 
tray  containing  xylol  or  toluol,  where  it  remains  until  the 
paraffin  is  dissolved.  It  is  then  transferred  to  a  tray 
containing  95%  alcohol.  In  the  course  of  a  few  minutes 
the  plate  is  taken  out,  and  the  alcohol  drained  off.  The 
sections  are  now  covered  with  a  thin  layer  of  solution  No. 
2,  after  which  the  plate  is  set  aside  until  the  photoxolyn  or 
celloidin  dries.  The  plate  can  now  be  placed  in  the  stain 
(Boehmer's  hsematoxylin  and  eosin  for  instance).  After 
staining,  washing,  and  dehydrating,  the  sections  are  cleared 
in  oil  of  origanum.  It  should  be  stated  that  in  the 
stains  or  in  the  water  used  for  washing  off  the  stains,  the 
thin  layer  of  photoxolyn  or  celloidin,  to  which  the  sections 
adhere,  separates  from  the  glass  plate ;  the  film  may  then 
be  taken  up  from  two  sides,  and  transferred  from  one  solu- 
tion to  another,  until  cleared  in  the  oil  of  origanum. 
With  a  small  pair  of  scissors  the  sections  can  now  be  cut 
out>  and  mounted  in  balsam. 


Methods  for  Staining. 


It  has  long  been  known  that  when  properly  hardened 
tissues  are  subjected  to  the  action  of  coloring  matters,  cer- 
tain elements  of  the  tissues,  even  certain*  parts  of  the  cells, 
show  greater  affinity  for  the  stain  than  otffers.  This  selec- 
tive action  noticed  in  so  many  stains,  warrants  the  place 
they  hold  in  histological  technic.  Of  the  great  number  in 
use,  a  few  of  the  most  trustworthy,  and  such  as  can  be 
most  easily  made  and  used,  are  here  given. 

HJEMATOXYLIN  SOLUTIONS. 
Boehmer's  Solution. 

This  solution  is  one  of  the  best  in  use,  and  is  made 
after  the  following  formula:  — 

Hsematoxylin  crystals         *         .         .         .1  grm.)       ,   T 
Absolute  alcohol          .        .      •  .  10  c.  c.j    ' 

Potash  alum        ,         .         .   .      .         .         10  grms .)       1   TT 
Distilled  water  '..'       .  200 ;  c.  c.\  .  S< 

The  crystals  of  hsematoxylin  are  dissolved  in  the  abso- 
lute alcohol,  and  kept  in  a  well-stoppered  bottle  for  24 
hours  (solution  I). 

The  alum  is  dissolved  in  warm  distilled  water,  aud  al- 
lowed to  cool,  keeping  it  free  from  dust  (solution  II).  Add 
solution  one  to  solution  two,  stir,  and  keep  in  an  open  dish 
for  about  a  week ;  filter,  and  the  solution  is  ready  for  use. 

The  tissues  must  be  stained  in  section ;  the  steps  are 
as  follows : — 

The  tissues  may  be  hardened  in  Miiller's  fluid,  alcohol, 
mercuric  chloride,  picric  acid,  nitric  acid,  etc.,  are 


—  152— 


I 

Embedded  in 

4 


v  v 

Celloidin.  Paraffin. 

Distilled  water.  Fix  to  slide  or  cover  with 

albumen  fixative  or  dextrin  solution. 

.  I 

Warm  until  paraffin  melts. 

V 
Dissolve  paraffin  in  toluol  or  xylol. 

Wash  in  absolute  alcohol. 

Wash  in  95%  alcohol. 

V 
Wash  in  distilled  water. 

;      , * 


Boehmer's  heematoxylin  solution  for  5  to  30  minutes. 

I- 

0.5%  potash  alum  solution  5  minutes.     This  develops  stain, 
and  acts  as  a  mortant. 


Wash  in  distilled  water. 


Dehydrate  in  alcohol. 

V 
Clear  in  oil  of  bergamot. 


V  V 

Celloidin  sections.  Sections  fixed  to  slide  or  cover. 

I  I 

Mount  in  balsam.  Pass  through  xylol,  and  mount  in  balsam. 

Ehrlich's  Haematoxylin  Solution. 

This  solution  can  be  kept  for  a  long  time,  it  seems  to 
improve  with  age. 


—153— 

Hsematoxylin  crystals,        .         .                  .  .  2  grms. 

Absolute  alcohol,         .         .         .         .         .  .  20  c.  c. 

Glycerine, .  100  c.  c. 

Distilled  water,            ......  100  c.  c. 

Absolute  alcohol,         .         .         .         .         .  .  80  c.  c. 

Glacial  acetic  acid,      .         .         .         .         .  •  .  10  c.  c. 

Potash  alum  to  saturation. 

Mix  the  distilled  water,  the  glycerine,  and  80  c.  c.  of 
the  absolute  alcohol,  and  the  10  c.  c.  of  glacial  acetic  acid. 
Dissolve  the  haematoxylin  crystals  in  20  c.  c.  of  absolute 
alcohol,  add  to  the  above  solution,  and  shake  well  for 
several  minutes. 

The  solution  so  obtained  should  have  a  reddish  color, 
and  is  now  to  be  saturated  with  the  alum ;  filter  at  the  end 
of  24  hours. 

Ehrlich's  hsematoxylin  needs  to  mature  from  1  to  2 
months  before  it  can  be  used.  Steps  for  staining  are  as 
follows : — 

1.  Sections  remain  in  the  stain  from  10  to  30  minutes 
(they  do  not  easily  overs  tain). 

2.  Wash  in  distilled  water,  dehydrate,  clear  in  oil  of 
cloves  or  bergarnot,  and  mount  in  balsam. 

Delafield's  Haematoxylin  Solution. 

The  formula  is  taken  from  Behren's  Tabellen. 

Hsematoxylin  crystals,       .....  4  grms. 

Absolute  alcohol, 25  c.  c. 

Ammonium  alum,      ......  52  grms. 

Distilled  water,           .         .         .         .         .         .  400  c.  c. 

Glycerine, 100  c.  c. 

Methyl  alcohol,          ......  100  c.  c. 

The  hsematoxylin  crystals  are  dissolved  in  the  abso- 
lute-alcohol, the  alum  in  the  hot  water;  as  soon  as  the 
alum  solution  cools,  add  the  haematoxylin  solution.  Allow 
to  stand  in  a  wide  vessel  from  3  to  4  days,  filter,  and  add 
the  glycerine  and  methyl  alcohol. 

This  hsematoxylin  solution  is  generally  used  for  stain- 
ing tissues  en  masse.  Before  using,  dilute  the  stain  5  to  10 
times  with  distilled  water.  The  tissues,  which  may  be 
hardened  in  Miiller's  fluid,  mercuric  chloride,  picric  acid, 
alcohol,  or  Flemming's  or  Hermann's  solution,  remain  in  the 


—154— 

stain  for  24  to  48  hours,  are  then  rinsed  in  distilled  water, 
and  placed  in  an  acid  alcohol  wash — 

Alcohol  (70%} 100  c.  c. 

and  hydrochloric  acid 3  to  5  drops, 

for  3  to  6  hours.  They  are  then  washed  in  flowing  water 
for  1  to  2  hours,  dehydrated,  and  embedded  in  paraffin  or 
celloidin.  Embryos  are  very  well  stained  by  this  method. 

Heidenhain's  Haematoxylin  Solution. 

Tissues  need  to  be  hardened  in  alcohol,  and  stained 
en  masse.  Gland  tissues  are  well  stained  by  this  method. 
Small  pieces  of  the  tissue  are  placed  in  a  1%  aqueous 
solution  of  hsematoxylin  crystals,  where  they  remain  for 
8  to  12  hours.  They  are  then  transferred  to  a  1%  aqueous 
solution  of  bichromate  of  potash  for  12  to  18  hours ;  in 
this  solution  the  tissues  become  jet  black.  Embed  in 
paraffin. 

Heidenhain's  Iron-Lack-Haematoxylin  Solution. 

The  tissues  are  to  be  hardened  in  alcohol,  mercuric  chlo- 
ride, Flemming's  or  Hermann's  solution,  or  in  the  osmium, 
platinum  chloride,  and  mercuric  chloride  mixture  suggested 
by  the  author.  They  are  then  embedded  in  paraffin.  The 
sections  must  be  cut  very  thin.  These  are  fixed  to  cover 
glasses  with  albumen  fixative,  which  is  best  done  by 
floating  the  sections  on  warm  distilled  water,  and  drawing 
them  upon  a  cover  smeared  with  a  thin  layer  of  the  fixa- 
tive. 

The  steps  are  as  follows  :  — 

Remove  paraffin  from  sections, 

•4 

Place  cover  glass  with  sections  fixed  to  it  in  a  3%  aqueous 
solution  of  ammonium  sulphate  of  iron. for. I  to  3  hours.: 

V 

Rinse  in  distilled  water  for  a  few  minutes. 

I 

Place  sections  into  a  saturated  aqueous  solution  of  hsema- 
toxylin  crystals  for  1  to  3  hours. 

; 

Rinse  in  distilled  water. 


Wiish  in  the  ammonium  sulphate  of  iron  solution  until  the 

black  clouds  cease  to  be  given  off;  the  section  should 

now  have  a  bluish-gray  color. 

I 

Eiuse  in  distilled  water. 

4 

Dehydrate. 

I 

Clear  in  oil  of  bergamot. 

v 
Pass  through  xylol. 

V 

Mount  in  balsam. 

In  sections  stained  in  iron-lack-hsetnatoxylin,  the 
details  of  the  nuclear  structure,  as  also  the  accessory 
nucleus,  are  very  clearly  brought  out. 

Weigert's  Haematoxylin  Solution. 

This  is  used  for  staining  the  central  nervous  system. 
The  tissues  need  to  be  hardened  in  Miiller's  fluid,  are  then 
transferred  without  washing  into  alcohol,  and  embedded 
in  celloidin.  Thecelloidin  block  is  placed  in  the  following 
solution  for  24  to  48  hours : — 

Acetate  of  copper  (saturated  aqueous  solution)  )  1 

Distilled  water  .         .  j- equal  pa 

The  block  is  then  washed  in  70%  alcohol  for  24  hours, 
and  may  then  be  sectioned.  (If  desired  the  sections  may 
first  be  cut,  and  then  placed  in  the  above  solution  of  acetate 
of  copper  for  24  hours,  and  washed  for  several  hours  in  70% 
alcohol.)  Sections  are  now  stained  in  Weigert's  hsema- 
toxylin  solution. 

Haematoxylin  crystals, 1  grm. 

Absolute  alcohol,          .         .         .         .         .         .  10  c.  c. 

Lithium  carbonate, 1.2  grms. 

Distilled  water, 100  c.  c. 

The  hsematoxylin  crystals  are  dissolved  in  the  abso- 
lute alcohol,  the  lithium  carbonate  in  the  water;  mix  the 
two  solutions.  The  sections  remain  in  the  stain  12  to  24 


hours,  are  then  washed  in  Weigert's  differentiating  fluid ; 

Borax,         .......         .2  grms. 

Potassium  ferricyanide,     .         .         .         .         .      2.5  grms. 

Distilled  water,  .;        .         .         .         .         .        100  c.  c. 

The  sections  remain  in  the  differentiating  fluid  until 
the  gray  matter  has  a  brownish-yellow,  and  the  white  a 
bluish-black  color.  Sections  are  then  washed  in  tap 
water  for  several  hours,  dehydrated  in  alcohol,  and  cleared 
in  the  following  clearing  fluid  (Weigert's)  : — 

Carbolic  acid  crystals         ...         1  part. 
Xylol  v 3  parts. 

The.  sections  may  then  be  mounted  in  balsam. 
This  method  has  been  variously  modified ;   two   such 
modifications  are  here  given. 

Pal's  Method. 

Prepare  and  cut  sections  as  for  Weigert's  stain.  Stain 
in  Weigert's  hsematoxylin  solution,  after  staining  wash  the 
sections  in  water  to'which  a  trace  of  lithium  carbonate  has 
been  added.  The  sections  are  then  placed  in  a  ^%  aqueous 
Solution  of  permanganate  of  potash  in  which  they  remain 
20  to  30  seconds,  are  then  rinsed  in  distilled  water,  and 
differentiated  in  the  following  solution  : — 

Oxalic  acid     ...         .         .         .         1  grm. 

Potassium  sulphite  (K2  So3)     .         .         1  grm. 

Distilled  water  .         .         .         .200  c.  c. 

They  remain  in  this  solution  for  i  to  1  minute,  or  until 
the  gray  matter  has  assumed  a  yellowish  color,  are  then 
washed  in  water,  dehydrated,  cleared  in  Weigert's  clearing 
fluid,  and  mounted  in  balsam. 

Benda's  Method. 

Bemla's    modification    is     usually    followed    in    this    laboratory. 
Harden  tissues  in  Miiller's  fluid. 

I 

Embed  in  celloidin. 

\, 

•    Sections  are  placed  for  12  to  21  hours  in  the  following  solution  : 
Liquor  ferri  ter  stilphatis          .        *        1  part. 
Distilled  water  .        .        .        .        2  parts. 


—157— 

I 
Rinse  thoroughly  in  two  tap  waters  and  one  distilled  water. 

I 

Stain  for  24  hours  in  -  - 

Hsematoxylin  crystals         .         .  1  grra. 

Absolute  alcohol          .        .        .  10  c.  c. 

Distilled  water  ...  90  c.  c. 

V 

AY  ash  sections  in  tap  water  for  10  to  15  min. 

V 

Place  in  0.25%  aqueous  solution  of  permanganate  of  pot- 

ash  until  the  gray  matter  begins  to  stand  out 

(1  to  15  minutes). 

4 

Wash  in  distilled  water. 

V 

Differentiate  in  the  following  solution  - 

Hydric  sulphite          .        .        .        about  5  to  10  c.  c. 

Distilled  water  ....  100  c.  c. 

until  the  gray  matter  has  a  light  yellow  color. 

I 

Wash  in  tap  water  for  1  to  2  hours. 

v 

Rinse  sections  in  distilled  water. 


Double  stain  in  alum  carmine  for  1  to  1^2  hours, 
wash  in  distilled  water. 


Dehydrate. 

4 

Clear  in  Weigert's  clearing  fluid. 

4 

Mount  in  balsam. 

If  sections  have  been  kept  for  some  time  in  alcohol,  it 
is  advisable  to  place  them  for  24  hours  in  Miiller's  fluid, 
from  which  they  are  rinsed  in  distilled  water,  and  stained 
after  any  one  of  the  above  three  methods. 


—158— 
CARMINE  STAINS. 

Carmine  has  for  many  years  held  a  prominent  place 
among  the  stains  used  for  coloring  tissue.  It  is  especially 
useful  for  staining  them  in  bulk. 

Grenadier's  Borax  Carmine  Solution. 

Carmine  (No.  40).  .         .         .         ..        .3  grms. 

Borax 4  grms. 

Distilled  water .        100  c.  c. 

Alcohol  (70%) '   .        100  c.c. 

The  carmine  and  borax  are  dissolved  in  warm  distilled 
water,  allowed  to  cool,  after  which  the  alcohol  is  added ;  at 
the  end  of  48  hours,  the  solution  is  filtered;  the  filtrate 
must  stand  for  several  weeks  before  using.  Tissues  hard- 
ened in  alcohol  or  bichloride  of  mercury  are  stained  well 
after  this  method.  The  pieces  remain  in  the  stains  from 
24  to  48  hours,  are  then  washed  in  an  acid  alcohol  wash  (6 
to  8  drops  of  HC1  to  100  c.  c.  of  70%  alcohol)  from  6  to 
24  hours,  and  then  in  70%  alcohol  for  two  hours,  dehy- 
drated, and  embedded  in  celloidin  or  paraffin. 

Grenadier's  Alum  Carmine  Solution. 

Carmine  (No.  40),      „       ,.  .      .         .         .         :         1   grin. 

Potash  alum,        .     .......       ...         .         .     3-5  grms. 

Distilled  water,  ...        ...         .       100  c.  c. 

Add  the  alum  and  carmine  to  the  water ;  place  over  a 
flame,  and  bring  the  water  to  the  boiling  point;  allow  to 
boil  15  minutes.  As  soon  as  the  solution  is  cold,  filter,  arid 
it  will  be  ready  for  use.  Tissues  hardened  in  the  chrome 
salts,  alcohol,  or  bichloride  of  mercur}^  are  well  stained  in 
this  solution ;  stain  en  masse.  The  pieces  remain  in  the 
stain  from  24  to  48  hours  (even  longer  if  they  are  large), 
are  then  washed  for  several  hours  in  flowing  water,  dehy- 
drated, and  embedded  in  paraffin  or  celloidin. 

Orth's  Lithium  Carmine  Solution. 

Carmine,  .         .         .         .  -.         2.5  grms. 

Lithium  carbonate,  .         .  .  ,»  .       .         1.2  grms. 

Distilled  water,         .         .         .      •   .         .         .  100  c.  c. 

The  carmine  and  lithium  carbonate  are  dissolved  in 
warm  water ;  allow  to  cool,  and  filter.  Sections  remain  in 


—159— 

the  stain  10  to  15  minutes,  are  then  washed  in  acid  alcohol 
or  in  70%  alcohol  to  which  a  few  crystals  of  picric  acid 
have  been  added ;  remove  acid  by  transferring  sections  to 
70%  alcohol.  Dehydrate,  clear  in  oil  of  bergamot,  and 
mount  in  balsam. 

Orth's  Picro-Lithium  Carmine  Solution. 

To  three  parts  of  the  above  lithium  carmine  solution 
add  one  part  of  a  saturated  aqueous  solution  of  picric  acid. 
Sections  stain  in  10  to  20  minutes.  Wash  in  70%  alcohol 
to  which  a  few  crystals  of  picric  acid  have  been  added. 

ANILINE  DYES. 

It  would  lead  far  beyond  the  scope  of  this  guide,  to 
mention  even  very  briefly,  the  various  aniline  dyes  that 
have  found  their  way  into  microscopical  technic.  The 
certainty  and  ease  with  which  many  of  the  microorganisms 
are  stained  by  them,  has  made  them  very  useful  in  bacter- 
iological work.  Their  use,  as  staining  agents  for  tissues, 
dates  back  to  1863,  at  which  time  Waldeyer  and  Frey  made 
use  of  one  of  the  fuchsins  for  staining  tissues.  Since  that 
time  an  ever  increasing  number  of  these  dyes  has  been 
tried,  and  recommended  by  various  investigators.  The  few 
here  mentioned  have  proved  themselves  most  useful  in  the 
hands  of  the  author. 

Ehrlich  classifies  all  aniline  dyes,  as  basic, 
neutral,  or  acid.  In  a  general  way  we  may  say  that  the 
basic  dyes,  such  as  methylene  blue,  methyl  green,  gentian 
violet,  methyl  violet,  Bismark  brown,  safranin,  etc.  are  to  be 
used  when  it  is  desired  to  stain  the  nucleus,  while  the  acid 
aniline  dyes,  such  as  eosin,  orange  G,  acidfuchsin,  lichtgrun, 
aurantia,  and  nigrosin  are  generally  used  for  staining  the 
protoplasm  of  cells. 

Almost  all  aniline  stains  are  readily  soluble  in  alcohol 
or  water,  and,  as  they  stain  very  deeply  and  quickly,  only 
dilute  solutions  (0.5  to  1%)  are  recommended.  The 
majority  of  the  basic  stains  give  the  best  results  when 
combined  with  aniline  ivater,  but  as  nearly  all  stain 
diffusely,  the  sections  need  to  be  thoroughly  washed  in 
water,  alcohol,  or  acidulated  alcohol  before  mounting. 


— 160 — 

Flemming's  Safranin  Solution. 

Safranin         .         .         .         .  1  grm. 

Absolute  alcohol  .         .         .          10  c.  c. 

Aniline  water         .         .         .         .         90  c.  c. 

The  aniline  water  is  prepared  by  shaking  5  to  8  c.  c. 
of  aniline  oil  with  100  c.  c.  of  distilled  water,  and  filtering 
through  a  wet  filter  paper.  Dissolve  the  safranin  in  aniline 
water,  and  add  the  alcohol.  Filter  before  using. 

The  steps  for  staining  are  as  follows : — 

Harden  tissues  in  Flemming's  or  Hermann's  solution. 

4 

Embed  in  paraffin. 

I 

Cut  very  thin  sections,  and  fix  to  cover  glasses. 

I 

Remove  paraffin  with  toluol  or   xylol,   and,   after  rinsing 
sections  in  alcohol,  transfer  into  distilled  water. 

I 

Stain  in  Flemming's  safranin  solution  for  24  to  48  hours. 

I 

Rinse  in  distilled  water. 
I 

v 

Wash  thoroughly  in  95%  alcohol  (200  c.   c.)  to 
which  1  or  2  drops  of  HC1  have  been  added. 

I 

Wash  in  alcohol,  clear  in  oil  of  bergamot,  pass  through 
xylol,  a'nd  mount  in  balsam. 

In  tissues  hardened  and  stained  in  the  above  way,  the 
chromatic  network  of  the  nucleus  is  well  stained ;  this 
method  is  largely  used  for  staining  cells  in  process  of 
division. 

Gentian  Violet    Solution  (Ehrlich  and  Bizzozero),     Taken 
from  Lee's  Vade  Mecum. 

Gentian  violet         .         .         .         .  .'  1  part. 

Alcohol 15  parts. 

Aniline  oil 3  parts. 

Distilled  water       ....  80  parts. 


Mix  the  aniline  oil  with  water,  and  filter  through  wet 
filter  paper.  Dissolve  gentian  violet  in  aniline  water,  and 
add  the  alcohol. 

The  steps  of  the  method  are  as  follows :  — 

The  tissues  are  hardened  in  Flemming's  or  Hermann's 
solution. 

4 

Embed  in  paraffin,  section,  and  fix  sections  to  cover  glasses  ; 

remove  paraffin  from  sections,  and  bring  them 

into  distilled  water. 

4 

Stain  in  the  above  solution  for  10  to  15  hours. 


Rinse  in  alcohol. 

V 

Wash  in  1%  aqueous  sol.  of  chromic  acid  30  seconds. 

4 

Wash  in  alcohol  for  a  few  seconds. 

I 

Again  bring  into  1%  aqueous  sol.  of  chromic  acid  30  seconds. 


Wash  in  alcohol  for  30  to  40  seconds. 

;-4 

Clear  in  oil  of  cloves;  the  oil  needs  to  be  changed  once   or 

twice,   as  much  of  the  stain  is  given  off  in  it. 

Sections  remain  in  the  oil  until  no  more 

stain  is  given  off. 

I 
V 

Pass  through  xylol,  and  mount  in  balsam. 

Tissues  hardened  in  alcohol  need  to  be  stained  only  10 
to  15  minutes  ;  the  after  treatment  is  the  same  as  above 
given. 

This  method  brings  out  clearly  nuclear  structure  and 
karyokinetic  figures. 

Methylene  blue,  methyl  violet,  methyl  green,  and  Bis- 
mark  Brown,  may  be  made  up  as  \%  solutions  in  about 
%  alcohol.  The  tissues  hardened  in  alcohol  and  mercuric 


chloride  will  be  stained  in  10  to  15  minutes  ;  tissues  hard- 
11 


— 162 — 

ened  in  Flemming's  or  Hermann's  solution  in  as  many 
hours. 

Wash  sections  thoroughly  in  alcohol,  and  mount  in 
balsam. 

As  the  acid  aniline  dyes  are  generally  used  for  counter- 
staining,  they  will  be  described  under  the  head  of  double 
and  triple  stains. 

DOUBLE  STAINING. 

When  certain  colors  are  combined  in  a  solution,  or 
used  one  after  the  other  in  staining,  it  has  been  found  that 
some  elements  of  the  tissues  to  be  stained  are  colored  by 
one  of  the  dyes  used,  while  others  show  greater  affinity  for 
other  dyes.  This  fact  is  made  use  of  in  combining  dyes 
for  double  and  triple  staining. 

Haematoxylin  and  Eosin  or  Acid  Fuchsin. 

The  eosin  solution  used  for  double  staining  is  made 
after  the  following  formula : — 

Eosin         .        V.V       .         .         .  ,      .  '      1  grm. 
Alcohol  (60%)          ..        .  ,      .        .     100  c.c. 
The  acid  fuchsin  solution — 
Acid  fuchsin     .       .  ."        .         .         1  to  2  grms. 
Distilled  water          .         »        .  100    c.c. 

The  steps  for  staining  are  as  fallows  : — 


-i63- 

Boehmer's  Ehrlich's  Delafield's 

Haematoxylin.  Hasmatoxylin.  Haematoxylin. 

Tissues  inny  be  hardened  in  alcohol,  Mtiller's 
fluid,  or  bichloride  of  mercury. 


Embed  iu 


v 
Embed  in 


Stain  en  masse  for  24 
to  48  hours. 


Par.-iffin.     Celloidin.     Parnffin.     Celloidin. 


1 

| 

Wash  in  acid  alcohol 

V 
Fix  sections  to 

v 
Fix  sections  to 

2—6  hours. 

cover  glass. 

cover  glass. 

1 
v 

I 

1 

In  flowing  tap  water 

Remove 

v 
Remove 

1  —  2  hours. 

paraffin. 

paraffin. 

]f 

U             U      ' 

r                    Dehydrate. 

y 

Distilled  water.             Distilled  water.                Embed  in 

v 

I                              1             1 
v                 Paraffin.       Celloidin. 

Stain  from  10  to  30             Stain  for  16  to  60              1 

minutes.                            minutes.           ™      v    ,. 
I"  ix  sections  to 

I 

cover  glass. 

V                                                     V 

Wash  in  0.5%  potash       Wash  in  tap  water 
alum  5  minutes.            10  to  15  minutes.       Remove 

| 

paraffin. 

v 

V                    i               v 

Distilled  water.              Distilled  water.             Distilled  water. 

V 

Stain  in  eosin  for  5  to  10  minutes,  or  in  acid  fuchsin 
for  10  to  15  minutes. 


Wash  in  distilled  water. 


Wash  and  dehydrate  in  alcohol. 

v 
Clear  in  oil  of  bergamot. 


Paraffin  sections  fixed  to 
cover  glass. 

v 

Pass  through  xylol,  and  mount 
in  balsam. 


V 

Celloidin  sections. 


Mount  in  balsam. 


Orange  G  and  Haematoxylin  (Rawitz). 

The  tissues  should  be  hardened  in  absolute  alcohol,  mercuric 
chloride,  or  picric  acid. 

4 

Embed  in  paraffin,  and  fix  sections  to  cover  glass. 

V 

Remove  paraffin,  and  bring  sections  into  distilled  water. 
I 

V 

Stain  in  a  saturated  aqueous  solution  of  Orange  G  (filtered  ; 
for  about  24  hours. 

I 

Rinse  in  distilled  water. 

j 

Stain  in   Boehmer's    haematoxylin    solution    for    about    5 
minutes. 

I 

Wash  in  distilled  water. 

I 

Dehydrate  in  alcohol. 

I 

Clear  in  oil  of  bergamot. 

V 

Pass  through  xylol,  and  mount  in  balsam. 

This  is  certainly  one  of  the  best  of  double  stains, 
especially  for  glandular  tissues.  The  nuclei  are  stained 
with  the  hsematoxylin,  the  protoplasm  with  orange  G. 

Safranin  and  Lichtgruen  (Benda). 

Tissues  hardened  inFlemming's  or  Hermann's  solution 
are  embedded  in  paraffin,  and  sections  fixed  to  the  cover 
glass.  Remove  paraffin,  and  bring  sections  into  distilled 
water. 

Stain  for  about  24*  hours  in  the  following  safranin 
solution : — 

Safranin 1  grm. 

Aniline  water          .          .         .  90    c.  c. 

Absolute  alcohol  .  10    c.  c. 


— 165— 

Sections  are  then  washed  in  absolute  alcohol  for  30 
seconds,  and  in  a  \%  alcohol  solution  of  lichtgriin  for  15 
to  45  seconds,  again  washed  in  absolute  alcohol,  cleared  in 
oil  of  bergamot,  passed  through  xylol,  and  mounted  in 
balsam. 

When  the  stain  is  properly  managed,  the  chromatic 
network  of  the  nucleus  and  the  chromosoma  of  dividing 
cells  are  stained  red,  the  protoplasm  green. 

Nigrosin  and  Eosin. 

This  method  is  useful  for  staining  sections  of  the  cen- 
tral nervous  system.  Sections  are  stained  for  one  hour  in 
a  \%  solution  of  nigrosin,  are  then,  washed  in  distilled 
water  for  several  minutes;  they  are  then  placed  in  a  1% 
solution  of  eosin  in  which  they  remain  from  3  to  5  minutes. 

Wash  first  in  70%  alcohol,  then  in  95X  until  no  stain 
is  given  off  from  the  section.  Clear  in  oil  of  bergamot,  and 
mount  in  balsam. 

Aniline  Blue  and  Safranin  (Stroebe). 

This  method  is  especially  to  be  recommended  for  stain- 
ing peripheral  nerves.  The  method  as  here  outlined  is 
slightly  modified  from  that  given  by  Stroebe. 

The  peripheral  nerves  are  hardened  in  Miiller's  fluid, 
embedded  in  paraffin,  and  cut  longitudinally  or  trans- 
versely. Sections  are  fixed  to  cover  glas  es,  the  paraffin 
removed,  and  carried  through  alcohol  into  distilled  water. 

1.  Sections  are  then  stained  for  1  to  5  hours  in  a  satu- 
rated aqueous  solution  of  aniline  blue  (Grtibler's  wasserlos- 
liches  anilin  blau). 

2.  They  are  then  rinsed  in  distilled  water. 

3.  Decolorize  in  an  alkali  alcohol  wash,   which  is  pre- 
pared by  dissolving  1  grin,  of  caustic  potash  in  100  c.  c.  of 
absolute  alcohol,  and   filtering.     Just  before  using,  about 
30  drops  of  this  solution  are  added  to  30  c.  c.  of  absolute 
alcohol.     On  placing  the  sections  into  the  wash,  they  loose 
their  blue  color,  and  assume  a  reddish-brown  tinge,  at  the 
same  time  reddish-brown  waves  are  given  off  from  the  prep- 
aration. The  bleaching  is  continued  until  the  reddish-brown 
clouds  cease  to  be  given  off,  usually  about  1  to  2  minutes. 


— 166— 

4.  The  sections  are  now  transferred  to  distilled  water, 
in  which  the  sections  agnin  assume  a  blue  color,  of  a  much 
lighter  hue,  however.     In  the  distilled   water  the  sections 
remain  about  10  minutes. 

5.  Counter-stain  in  a  saturated  aqueous    solution    of 
safranin  for  about  30  minutes. 

6.  Rinse  in  distilled  water. 

7.  Wash,  and  dehydrate  quickly  in  95%  and  absolute 
alcohol. 

8.  Clear  in     oil  of  bergamot,   pass    sections    through 
xylol,  and  mount  in  balsam. 

The  axis  cylinders  are  all  stained  blue,  the  myelin  a 
yellowish-red  or  orange  color,  and  all  nuclei  red. 

Carmine  and  Aniline  Blue  (Duval). 

Tissues  are  stained  in  borax  carmine  in  the  usual  way, 
{ire  then  embedded  in  paraffin,  sectioned,  and  sections  fixed 
to  cover  glasses.  Remove  paraffin  from  sections,  and  bring 
them  into  absolute  alcohol. 

Stain  for  10  to  20  minutes  in — 

Aniline  blue* saturated  alcohol  solution   10  drops. 

Absolute  alcohol         .         .         .         .          10     c.  c. 

Wash,  and  clear  sections  in  turpentine,  puss  through 
xylol,  and  mount  in  balsam. 

The  central  nervous  system  is  well  stained  after  this 
method;  the  tissues  may  be  hardened  in  bichloride  of 
mercury.  The  connective  tissue  elements  are  stained  blue, 
the  nerve  cells  and  axis  cylinders  a  reddish-violet. 

Borax  Carmine  and  Indigo  Carmine  fNorris  and  Shakes- 
peare, taken  from  Bobm  and  Oppel's  Tashenbuch 
der  inikroskopischeft  Technic). 

Sol.  A.  Grind  in  a  mortar. 

Carmine       .         .         .         .         .         .         2  grms. 

Borax  .         .         .         .         .         .         8  grms. 

Distilled  water  .         .         .         .     130     c.  c. 

Allow  to  stand  for  24  hours,  then  filtor. 

Sol.  B.  Rub  up  in  mortar. 

Indigo  carmine  .....         8  grms. 

Borax '  8  grms. 

Distilled  water  .         .         .         .     130    c.  c. 

Allow  to  stand  for  24  hours,  then  filter. 


-i67- 

Before  using,  mix  equal  parts  of  sol.  A  and  B.  Stain 
tissues  for  20  minutes,  wash  in  a  concentrated  aqueous  sol. 
of  oxalic  acid,  rinse  in  absolute  alcohol,  dehyrate,  and 
mount  in  balsam. 

Tissues  may  be  hardened  in  Miiller's  fluid,  thoroughly 
washed  in  water,  and  embedded  in  celloidin. 

TR  IPLE  STAINING. 

The  formula  here  given  was  first  recommended  by 
Ehrlich,  and  used  by  him  for  staining  blood  preparations. 
Biondi  and  Heidenhain  have  slightly  modified  it,  to  make 
it  more  suitable  for  section  staining. 

Acid  fuchsin  (sat.  aqueous  sol.)  .  20  parts. 
Methyl  green  (sat.  aqueous  sol.)  .  50  parts. 
Orange  G  (sat.  aqueous  sol.)  .  100  parts. 

The  acid  fuchsin  and  orange  Gr  are  mixed,  the  methyl 
green  is  slowly  added  while  constantly  stirring.  The 
stain  should  not  be  filtered,  and  needs  to  be  diluted  with 
distilled  water  40  to  60  times  before  using. 

Steps  for  staining  are  as  follows  :  — 

Harden  tissues  in  absolute  alcohol  or  bichloride  of  mercury. 

I 

Embed  in  paraffin. 

I 

Fix  sections  to  cover  glasses. 

\, 

Remove  paraffin,  and  bring  sections  into  distilled  water. 

v 

Stain  for  4  to  24  hours. 

I 

Rinse  in  distilled  water. 


Wash  thoroughly  in  alcohol. 

V 
Dehydrate,  and  clear  in  oil  of  bergamot. 

I 

Pass  through  xylol,  and  mount  in  balsam. 


-i  68— 

Griibler  of  Leipzig  sells  the  stain  in  the  form  of  a 
powder.  A  \%  aqueous  solution  is  made  of  the  powder, 
but  before  using,  it  is  neccessarry  to  add  6  to  10  drops  of  a 
sat.  aqueous  sol.  of  acid  fuchsin.  Use  as  above  directed. 

The  nuclei  are  stained  a  bluish-green,  the  protoplasm 
a  reddish-purple,  fibrous  connective  tissue  f uchsin-red,  and 
the  red  blood  cells  orange. 


Methods  for  Preparing  and  Staining  Blood 
Preparations. 

The  steps  for  obtaining  blood  preparations  are  as  fol- 
lows : — Arrange  on  a  piece  of  filter  paper  a  number  of 
carefully  cleaned  cover  glasses ;  these  must  be  very  thin, 
extra  number  one.  Ehrlich  washes  the  cover  glasses  first 
in  strong  sulphuric  acid,  rinses  in  water,  and  places  them 
for  a  few  moments  in  glacial  acetic  acid ;  they  are  then 
washed  in  flowing  water  until  all  the  acid  is  removed, 
transferred  to  95%  alcohol,  from  which  they  are  taken  and 
wiped.  Unless  the  cover  glasses  are  thin  and  clean,  no 
good  preparation  can  be  made.  The  finger  is  pricked  with 
a  steel  pen,  one  of  the  prongs  of  which  has  been  broken 
off.  From  the  flowing  blood  a  very  small  drop  is  caught 
on  the  cover  glass,  near  its  edge,  and  the  glass  quickly 
placed,  blood  side  downward,  on  another  cover  glass,  care 
being  taken  to  cover  this  second  cover  glass  only  about 
half.  The  blood  will  be  seen  to  spread  out  between  the 
two  covers.  Quickly  draw  one  cover  glass  from  the  other ; 
a  thin  layer  of  blood  will  in  this  way  be  spread  on  both 
slips.  Ten  to  twenty  preparations  are  to  be  made  in  this 
way,  and  placed,  blood  side  up,  on  the  filter  paper  before 
you,  and  allowed  to  dry.  Before  staining,  the  preparations 
need  to  be  fixed.  This  is  done  by  one  of  the  following  two 
methods.  You  may  harden  the  preparations  in — 

Ether  .         .         .         .         ...         3  parts. 

Absolute  alcohol  ....         1  part. 

In  this  solution  they  remain  for  1  to  24  hours,  are  then 
set  aside  for  a  few  moments  until  the  ether  and  alcohol 
evaporates,  when  they  are  ready  for  staining. 


— i7o— 

Ehrlich  recommends  the  fixing  of  the  preparations  by 
heat.  He  suggests  a  very  simple  apparatus,  shown  in  Fig. 
II,  by  means  of  which  the  blood  may  be  fixed. 

It  consists  of  a  copper  plate  about  15  inches  long,  4 
wide,  and  -J  inch  thick.  The  copper  plate  is  heated  at  one 
end  by  means  of  an  alcohol  or  gas  flame.  If  then,  at  the 


»oo. 


Fig.  II.     Plate  for  heating  blood  preparations, 
blood  preparations;  F,  tripod. 


A,  copper  plate;    C, 


end  of  15  minutes  a  glass  rod  which  has  been  dipped  into 
water  be  passed  over  the  plate,  beginning  at  the  end  away 
from  the  flame,  a  place  is  reached  where  the  water  begins 
to  boil ;  this  region  of  the  copper  plate  is  looked  upon  as 
having  a  temperature  of  100°  C.;  it  is  represented  by  a 
dotted  line  in  the  diagram.  The  blood  preparations  (C) 
are  placed  on  the  plate  (blood  side  up)  between  the  flame 
and  this  imaginary  line  (nearer  the  latter),  and  heated  for 
a  time  varying  with  the  stain  to  be  used. 

The  blood  of  any  vertebrate  may  be  spread  and  fixed 
as  above  directed.  When  desired  to  make  preparations 
from  fish,  amphibian,  or  reptilian  blood,  the  blood  is  most 
easily  obtained  from  the  heart.  This,  after  exposure,  is 
nipped  with  a  pair  of  scissors,  a  small  drop  of  the  flowing 
blood  is  picked  up  on  the  end  of  a  clean  glass  rod,  quickly 
transferred  to  cover  glass,  and  spread  in  the  usual  way, 
Bird's  blood  is  readily  obtained  from  one  of  the  toes,  which, 
after  anaesthetizing  the  animal,  may  be  cut  or  partially 
amputated  ;  a  small  drop  is  taken  from  the  flowing  blood, 
and  spread.  Preparations  of  red  bone  marrow,  of  the  spleen, 
or  of  lymph  glands  may  also  be  prepared  after  this  method. 
Very  small  pieces  of  the  tissue  are  placed  between  two 


cover  glasses  and  crushed,  after  which  procedure  the 
covers  are  quickly  drawn  apart;  or  the  cover  glasses  may 
l»e  drawn  over  the  freshly  cut  surface  of  the  tissue.  Ihese 
preparations  may  after  fixing  be  stained  after  any  one  of 
the  following  methods  : — 

Haematoxylin  and  Eosin  Stain. 

The  preparations  are  fixed  for  about  1  hour  in  ether 
and  alcohol  or  are  exposed  to  a  temperature  of  100°  to  110° 
C.  for  about  45  to  60  minutes,  stained  in  Boehmer's  hsema- 
toxylin  for  20  to  30  minutes,  washed  in  flowing  water, 
counter-stained  in  eosin  for  5  minutes,  again  washed,  and 
quickly  dried  between  filter  -paper.  Mount  in  balsam. 

All  nuclei  are  stained  blue,  protoplasm  and  eosino- 
phile  granules  red.  The  other  granules  are  not  stained. 

Ehrlich's  Neutropkile  Mixture — 

Orange  G  (saturated  aqueous  solution),       .       130-135  c.  c. 
Acid  fuchsin  (saturated  aqueous  solution),         80-165  c.  c. 
Methyl  green  (saturated  aqueous  solution),  125  c.  c. 

Distilled  water,    '.  -:'     .  t       .         .         .         .  300  c.  c. 

Alcohol  (absolute),       .....  200  c.  c. 

Glycerine,     .         ....         .         .         .  100  c.  c. 

Mix  orange  G,  acid  fuchsin,  water,  and  absolute  alcohol 
in  a  bottle,  add  slowly  and  while  shaking  the  methyl 
green.  The  glycerine  is  then  added.  For  staining  in 
Ehrlich's  neutrophile  mixture,  the  preparations  need  to  be 
hardened  in  ether  and  alcohol  for  about  one  hour,  or  fixed 
at  a  temperature  of  100°  to  1105C.  for  15  to  30  minutes. 
Float  the  preparation  on  a  small  quanitity  of  the  stain  for 
about  15  minutes,  wash  in  flowing  water,  dry  between 
several  filter  papers,  and  mount  in  balsam.  The  red  cor- 
puscles should  have  a  reddish-brown  color  (brick  color), 
all  nuclei  green,  the  eosin ophile  granules  red,  and  the 
neutrophile  granules  violet-red. 
Chenzinski's  Stain. 

Methylene  blue  (saturated  aqueous  solution),  .  40  parts. 
Eosin  (1%  sol.  in  70%  alcohol),  ...  20  parts. 

Distilled  water, 30  parts. 

Glycerine,  10  parts. 


—  172  — 

The  eosin,  distilled  water,and  glycerine  are  mixed  in  a 
bottle;  the  methylene  blue  is  added  slowly  while  shaking. 
Preparations  need  to  be  fixed  from  1  to  1-J-  hours  at  a  tem- 
perature of  120°  C.  or  in  ether  and  alcohol  for  1  to  2  hours. 
They  remain  in  the  sta,in  for  24  hours  in  the  warm  oven  at 
a  temperature  of  40°  C.  Wash  quickly  in  flowing  water, 
dry  between  filter  paper,  and  mount  in  balsam.  The  red 
corpuscles  and  the  eosinophile  granules  are  stained  red,  all 
nuclei  blue. 

Ehrlich's  Triacid  Glycerine  Mixture. 

Aurantia,         ...       ..  •       .k        -.      •  .  ,  2  grms. 

Eosin      .         ;,         ...        .      •   .        .,_  •   v  3  grms. 

Nigrosin,         .         v        . '.       .         .  -  .  5  grms. 

Glycerine,      a      ....".        ..         .  .  40    c.  c. 

The  glycerine  is  divided  into  three  parts,  to  each  is 
added  one  of  the  above  stains,  and  each  needs  to  be  ground 
in  a  mortar  for  several  hours.  The  three  glycerine  solutions 
are  then  mixed,  and  exposed  to  a  temperature  of  60°  C.  for 
two  weeks.  The  stain  is  then  ready  for  use,  and  if  well 
made  amply  repays  all  the  trouble  taken  in  making.it.  It 
will  keep  for  a  long  time,  and  should  be  of  a  syrupy  con- 
sistancy^  The  blood  preparations  need  to  be  fixed  at  a 
temperature  of  130°  to  140°  C.  for  1  to  3  hours.  A  small 
quantity  of  the  stain  is  spread  out  in  a  flat  dish;  on  this 
the  preparations  are  placed,  they  remain  in  the  warm  oven 
(40°  C.)  for  24  hours;  are  then  washed  in  flowing  water, 
dried  between  filter  paper,  and  mounted  in  balsam.  The  red 
blood  cells  are  stained  yellow,  nil  nuclei  black,  the  eosino- 
phile granules  red,  other  granules  are  not  stained. 

Ehrlich's  Methylene  Blue  Solution. — This  is  employed 
for  staining  basophile  cells  (Mastzellen  of  Ehrlich). 
Methylene  blue  (saturated  alcoholic  solution),     .     1  part. 
Distilled  water,  .  .          ....     2  parts. 

Preparations  are  fixed  at  a  temperature  of  110°  C.  for 
30  minutes.  Stain  for  15  minutes.  Wash  quickly  in  flow- 
ing water,  dry  between  filter  paper,  and  mount  in  balsam. 
All  nuclei  are  stained  blue,  and  only  the  basophile  granules, 
which  also  take  a  blue  color,  are  stained. 


—  173— 

The  methods  here  given  are  especially  useful  for 
studying  the  blood  clinically,  and  can  not  be  too  warmly 
recommended. 

COUNTING  BLOOD  CELLS. 

The  instrument  now  generally  used  for  this  purpose  is 
the  Thoma-Zeiss  hsemacytometer.  This  apparatus  consists  of 
two  parts,  pipettes  by  means  of  which  the  blood  is  diluted 
100  times,  when  counting  red,  or  10  times  when  white 
blood  cells  are  to  be  counted  :  and  a  glass  slide,  on  which 
there  is  a  small  well  of  known  depth,  the  bottom  of  the  well 
being  divided  off  into  small  squares.  The  pipette  used 
when  counting  the  red  cells,  consists  of  a  capillary  tube, 
,  near  the  middle  of  which  there  is  an  ampullar  enlargement. 
This  is  so  graduated  that  the  cubical  contents  of  the  capil- 
lary tube  is  just  one-hundredth  part  of  the  cubical  con- 
tents of  the  ampulla.  The  blood  to  be  examined  is 
drawn  into  the  capillary  tube,  to  a  line  marked  1  (just  be- 
low the  ampulla),  the  end  of  the  pipette  is  then  inserted 
into  the  diluting  fluid,  and  this  is  sucked  up  until  the  di- 
luted blood  reaches  aline  marked  101  (just  above  the  am- 
pulla). The  pipette  is  then  carefully  shaken  to  mix 
thoroughly  the  blood  and  the  diluting  fluid. 

Either  of  the  following  two  solutions  may  be  used  for 
diluting  the  blood  : — 

Hayem's  Solution. 

Bichloride  of  mercury,  ...  0.5  grm. 
Sodium  chloride,  .  .  .  .  1.0  grm. 
Sodium  sulphate,  ....  5.0  grms. 

Distilled  water          .-*--     .         .         .  200.0     c.  c. 

Toison's  Fluid  (as  given  by  Kahlden). 

Methyl  violet  5  B,       .         .....'       .     0.025  grms. 

Neutral  glycerine,        .         .         .         .        30.0  c.  c. 

Distilled  water,  ....        80.0  c.  c. 

Mix  methyl  violet  in  glycerine  and  the  distilled  water; 
to  this  solution  is  added 

Sodium  chloride  (C.  P.),         .         .         1.0  grms. 

Sodium  sulphate  (C.  P.),        .         .         8.0  grms. 

Distilled  water,  .         .         .       80.0    c.  c. 


—174— 

Filter,  and  the  solution  will  be  ready  for  use.  The 
white  blood  cells  are  stained  violet,  and  may  thus  be 
counted  with  the  red. 

The  diluting  fluid  contained  in  the  capillary  tube  is 
then  blown  out,  and  a  small  drop  of  the  diluted  blood  is 
placed  on  the  centre  of  the  small  glass  disc.  This  small 
disc  is  surrounded  by  a  ring  of  glass,  cemented  to  the  slide. 
The  glass  ring  is  0.1  m.  m.  thicker  than  the  glass  disc. 
When  this  small  moist  chamber  is  covered  with  a  thick 
cover  glass,  we  have  a  layer  of  blood  0.1  m.  m.  deep  be- 
tween the  disc  and  the  cover  glass.  On  the  upper  surface 
of  the  small  glass  disc  (on  which  the  drop  of  diluted  blood 
was  placed)  there  are  marked  off  400  small  squares.  The 
sides  of  the  small  squares  are  ^  of  i  m.  m.  long.  It  will 
be  seen  that  the  layer  of  blood  over  each  of  the  squares 
would  have  a  cubical  contents  of — 

4oW  of  a  c.  m.  m.  (¥'T  x  ^  x  TV  =  ¥^). 

The  haemacytometer  slide  is  now  placed  on  the  stage  of 
the  microscope,  where  it  should  remain  undisturbed  for 
several  minutes  before  counting.  The  red  blood  cells  in 
25  to  50  squares  are  then  counted.  To  ascertain  the  num- 
ber of  red  cells  in  a  cubic  millimeter  the  following  formula 
may  be  useful : — 


-175— 


O    O 

•     PJ 


i.1 


— 176— 

Or  ascertain  the  average  of  the  red  blood  cells  in  the 
squares  counted,  and  multiply  this  number  by  400,000. 

In  case  it  is  desired  to  count  only  the  white  blood  cor- 
puscles, a  -J  %  sol.  of  glacial  acetic  acid  is  used  for  diluting 
the  blood.  This  solution  bleaches  the  red  cells,  and  brings 
out  clearly  the  white  corpuscles. 

The  blood  is  diluted  only  10  times,  using  for  this  pur- 
pose, the  Thoma-Zeiss  pipette  for  counting  white  corpus- 
cles. The  formula  then  reads  as  follows : 

(  The  number  of  white  ) 
4000  x  d  ( 10)  x  n  -  l.lood  co  r  p  u  s  c  1  e  s  [• 

( counted.  )         The  number  of  \vhite 

— 1=  blood    (tells    found  in 
n  sq  (Number  of  squares  counted).  cubic  millimeter. 

Or  multiply  the  average  number  of  white  corpuscles 
in  each  square  by  40,000. 


PLATE  I.— Blood  Elements. 

a,          Red  blood  cellsj  Erythrocytes,  stained  in  Ehrlich's  neutro- 

phile  mixture. 

a1 -a2,  Red  blood  cells  stained  in  eosin-methylen  blue. 
n,          Microcytes. 
h,          Poikylocyte. 
kL- k*,  Nucleated  red  blood  cells. 


6,          Polynuclear  white  blood   cells,   stained  in    eosin-methylen 

blue. 
i,  Polynuclear  white  blood  cells,  stained  in  Ehrlich's  neutro- 

phile  mixture. 

d,          Transitional  white  blood  cells. 
c1 ,         Mononuclear  white  blood  cell. 
c-e        Lymphocytes. 
/,  Eosinophile  white  blood  cells. 

<;,  Myelocytes,  stained  in  Ehrlich's  neutrophile  mixture. 

m,         Myelocytes  stained  in  methylen  blue. 
I,  Basophile  white  blood  cell, 

o,  Blood  plaques  or  blood  plates. 


PLATE  I. 


:•"-"'• 


o 


Q 


. 


' 


MEMORANDA. 


178  MEMORANDA. 


MEMORANDA.  179 


180  MEMORANDA. 


MKMORAXDA. 


MEMORANDA. 


MEMORANDA.  183 


ERRATA. 

Wherever  Fleming  appears,  read  Flemming. 
AVherever  Haidenhain  appears,  read  Heidenhain. 
Page  41,  seventh  line  from  bottom,  for  Schultz's  read 
Page  174,  read  ¥I5Vo-  of  a  c.  m.  in.   (F'ff  X  ^V  X 
stead  of    (       x         x    1 


INDEX. 

PAGE. 

ACCKSSOUY  nucleus,        -    .      ..     ;    .       ..     ^-.  •',./....     ,  ,„•  3 

Adenoid  tissue,          . .      .  '.    ?•  .  '      .         .    '*  .        ..  ;•',.  .         ,  67 

"      reticulum,          ...  -  •'.*..     .        .      '  •-_    -        •  68 

Adipose  tissue,            .        .        .      .  .        ...       «       "•        •   -•'.•••  26 

Albumen  fixative,               . ..;  •".  .....'     .    •  •' .  147 

Alcohol,  Ranvier's,  for  macerating  tissue,      ..        .-.;.-".  125 

"       95%",  for  hardening  tissues,        .        .        /       .      ,..••.  128 

Alum  carmine,             .        .        ....        .    ^  Y.       -V.v  158 

Amoeboid  movement  of  white  blood  cells,       ••••"'  .  .      .'      ".  -  7 

Aniline  blue  and  carmine,  double  stain,       .    .       " -.       -.-.    ,^-.-'t  .166 

"         "       "     safranin,        "         "                .      -,.       '•',     '   .  165 

Aniline  dyes,       .        .  "'    -.        .       .....  •  ,1  *      .    /" .  159 

"        "       acid,      .        .        .        .  .  ;\ .      .        .        .    •  ".  159 

basic.     .        .  '     .    . '-.        ;  •      .        .       \       \  159 

"         "       solubility  of,         ..        .   '     .        .        .    •  ,'v .-'•    =  .-.  159- 

Aniline  water,              .        ......        .        .     •-..•       -..  •  160 

Aorta,           .       s.       •        •     .    •    ..    •        •  •.    •  .  •.'..-•  ;-.   ,  •' ^     •  64 

Arterioles,        .^  ,.    .    .        .        .    .  ..        .        .      '..".;':..,  63 

Artery,     ..    .     ,  *.."  ,.    .    '     .     .  -. .    .    .         ..     '    .         .         .- :     V  63 

BENDA'S  hardening  fluid,            .        .        \              ',       "•>      ;..  130 

Benda's  modified  Weigert's  haematoxylin,       .        .        .      .- .  156 

Berlin  blue  injection  mass,         .         .    •    .         .   -;    .         .         »  139 

Bichloride  of  mercury  for  hardening,        .     -  >      ''•'  *    *        v  128 

Bile  capillaries,           .        .        .    >.        .        .        .  •  '     jt        .    .  98 

Bismark  brown,           .        .  .•     .        .       ..        »   ;    .        .        .  161 

Blood,  amphibian,      .     .   .        .        .•      ...        .         .        .'        .  6 

"       human,             .     "~  v        ."""    .        •        •         *  ;     •        •'  ^»  6 

"      pigeon,             .        .        .        .        ..,-..'       .        .  7 

"      stain,  detection  of,           .        .        .,       .        .        .        <4  10 

Blood  cells,  counting  of,     .        .        .        .        .    • '  .r        .       ,.-'  173 

Blood  preparations,  hardening  methods,          .   -     .      -:*•        169,  170 

"              "              stains;        .        .        .        ...'.%  171 

Chenzinski's,        .        .        .        .       ".        .        .        V     •'•'_  171 

Ehrlich's  methylene  blue,  .        .        .  "     .        .        .        .  172 

neutrophile,          .    ,,..  ...        .        .        .  171 

triacid •  .'        .  172 

Hsematoxylin  and  eosin,     .......  171 

Boehmer's  hsematoxylin,            .        .        .        .        .        .        .  151 

Bone,            .        .        .        .        .        .        .        .               .  *        .  33 


186  INDEX. 

Bone,  cancellated,       .        .^-      .".-'      ..     ;.  •     .    .    .     ".:;   .  37 

"      decalcified,        .        . ;'     >  •      .     ...      .     ->;       /y.  34 

"      developing,       .        .        ,        .      ".      -  .        .        .  ,      .  37 

"      marrow,     .         .        .         .     .    .        •.        .        .         .         .  38 

Borax  carmine,         .   .•      *        .         .    v  .        .         .         .     ••-'.  158 

"            "          and  indigo  carmine,  double  stain,           .        .  166 

Brain,           .    ;    :.        .        ','       .        -.        .     :  .        .        .         .  58 

Brunner's  glands,       .        .        ; 91 

CAPILLARIES,       ....        .         .        .        .        .        .        .  63 

Carmine  gelatin  injection  mass,        ....        .        .        .  138 

Carmine  stains ;          ....        .         .        .        .     •    .        .  158 

alum  carmine,       .'••'.' 158 

borax  carmine,             ........  158 

lithium  carmine,           . 158 

picro-lithium  carmine, 159 

Cartilage,             29 

elastic  fibro-, .        .        .  29 

hyaline,               ....'...  29 

white  fibrous,     .         .         .     .  -.      . --.       ..      :  .         .  30 

Caustic  potash,  for  macerating  tissues,    .                 .        .        .  125 

Cells,  animal, 2 

11      plant,         .        .        .        ..     ;. .       .        .        .        .        .  1 

Cerebellum, 58 

"           Golgi  stained,        .         .         .        .                 .        *.  54 

Cerebrum,  Golgi  stained, 54 

"       Weigert  stained,      .        .        .        .        .        .  59 

Chenzinski's  blood  stain, 171 

Ciliary  body, \-       '•£  ••  •  116 

Cleaning  cover  glasses  and  slides,             147 

Cochlea, .•.  .  .        .»      ,        .  117 

Connective  tissue, .,  J    .  21 

"               "        areolar,        .        .     v  .  .  .v.        ...  22 

cell  spaces,       ;  v  [•*        .        .        :        .  22 

*'                "       embryonic, 25 

"                "        white  fibrous,       .     .    .        ...        .        .  21 

"               "       yellow  elastic,     .        .        .        ....  21 

Cornea,        .        ...        „        .        .     .   ,        .        .        .  115 

Corpus  luteum,           .        .        .        .        .        t  ;     .        *.       .  Ill 

Corti,  organ  of,            .        .*     .      ..-     L      .       -.  -      .        ,  118 

Counting  blood  cells,          .        ;        ...        .        ...        .  173 

Cutting  sections ; 

celloid in  sections,         .        .      -  .    V    .        .-       .        .        .  142 

free  hand  sections,       .        .* 140 

freezing  method,           ........  141 

paraffin  sections,                .  .        .        -» .  •    ...r      ;"f '•  \  • . /..-....  147 

DECALCIFYING  methods,      .        .        .        .        .        .,     ..        .  132 

Ebner's         .         .         .       ' ..        ;        .,       .        .    i,    .         ..'  132 

Haug's      , .     .    .        .  133 


INDEX.  187 

Lepkowski's, 133 

nitric  and  hydrochloric  acid,       ......  132 

Delafield's  ha?matoxylin, .     '."..  153 

Dennis, .     ;  .  73 

Dextrin  method  of  fixing  sections  to  slide  or  cover,        .        .  149 

Double  staining, 162 

aniline  blue  and  carmine,           .        ...      .        ,        .        .  166 

aniline  blue  and  safranin,            .        ...,.„        .        .  165 

borax  carmine  and  indigo  carmine,            .      .,.        ;        .-  166 

hamiatoxylin  and  acid  fuchsin,           •  '.-•.      '•        .         162,  163 
lueinatoxylin  and  eosin,              .        ,        .        „        .         162,163 

hsematoxylin  and  orange  G,        .        .        .        .        »        .  164 

EBNER'S  decalcifying  fluid,        .        .        .      -'..'     "V        .        .  132 

Ehrlich's  hsematoxylin,               .      •..»-       i        .      •  V       .      '.  152 

methylene  blue  mastzellen  stain,  •   .        .      ..        .  172 

neutrophile  stain,      .  '•  .     '-  .        ,  ;"  -.     •   *        .  171 

triacid  stain,       .       '.      •  ,        .      :'-.f        .      ...  ]72 

Ehrlich-Biondi  triple  stain,       .       ',     ".      *t        ,        '.        .  167 

Ehrlich  and  Bizzozero's  gentian  violet  sol.,  ;  4     .-"-;        ,        .  160 

Embedding  tissues,             .      ;.      -.        f      r,     -.     ;'.        ,  140 

celloidin  or  collodiuni,      •    »      .  .        .        ,        ,        ;      :  ,  141 

gum  arabic,        ;'.        .        .        ,        ,     •   .     .;   .  ,      ..       .  141 

paraffin,        ..      .       ...        ,        .     '    ,"        f       >,       '.      '.  144 

Eudothelium,      .        .        .-     '.        ,f       .v  "     .        .      •  . .    >  *   '  26 

Epidermis,  macerated,       .        .        ....        ."        .       .'.  • '<  73 

Epididymis,         .        .        ,        .        .        .      :  ,'       ^        .        .  109 

Epithelium,      '  .       '.        ,        ,        .'      i      '  .'    •*-..*'       .        .,'  13 

ciliated,  teased,      ^       ,        «        .        .-   %  .      •'-.;  is 

section  of,        ..        f      ;/    ;  .     -  .      :  .'  18 

columnar,  teased,           .        ,        .        «        .-     ;1i  17 

"           section  of,     .        .  ,   .  .    •    .        .        .  17 

stratified  pavement,  teased,      •    .    -    .        .        .  13 

11               "          section  of,      .        .        .    .    .  14 

transitional,  teased,       .        .        .4      .        .        .  14 

"           section  of           .        .        .       ;.  ;   .  .  14 

Eye,           ^.'      .        .       "T'r  .     ',        .        .,    •  .        .     ••' .•  115 

FAT  cells,             .        .        ...     v  .•..,.        .  26 

Fibrin,               •  .        .        .*       .      /.      '  •*        .        »        ...  9 

Filiform  papillae,        'i        .        ...        „•  '      .        .        .  77 

Fixing  sections  to  slide  or  cover  glass,  methods  of,        .        .  147 

albumen  fixative,         .        ,f      ..   "•  ';        .       '',       \        .  147 

dextrin  method,        1',  •      .  •        •        •        *   m '   •        •  t   •  149 

Gaule's  method,           . 149 

Huber's  method,          .        v 148 

Flemming's  hardening  solution, 130 

Flemming's  safranin  solution, 160 

Fungiform  papillae, »        .        .        .  78 


188  INDEX. 

GAULE'S  method  of  fixing  sections  to  slide  or  cover,        .        .*••,  1,40 

Gentian  violet  solution,      .      •  .         .       ••       -•         .         .         .  100 

Goblet  cells,       '.         .      -.-       .•''.'•'    .         /    ' : . ' -.    v>. ;'...'  18 

Gold  chloride,  Ranvier's  method, ...         ....  ,      .   .    -,   .  ,    130 

Golgi  method,     ,        .    "...       v       v  •      .  -•-.-'..       .    ;   £$**.  135 

H/EMACYTOMETER,           .            .            ...            .        '    .      '•'':.          J ,    ^ '"  ;i  173 

Hamiatoxylin  solution,       .         . .  '    ;.         .      '-'¥'!-    '  .    '*-.•".  151 

Hrematoxylin  stains,          *.     '•  .      •  .         :    •  :  v        .        ••"''.•  151 

Boehmer's  solution,     .         .               '  ."       . :  :':  ^'' V  ;.; V  ''.  151 

Benda's  method,           .         .  .      t        ,     •     .•:     i  ;      ;-,•      .'-;  15(> 

Delafield's  solution,     .         .        ;         .  •  ••« .';/     .         /    '    ;  153 

Ehrlich's  solution,        .     "'-..•'•   ..       ., '       '.  -;  .  -* ;.-  -  v  •.'•• .- .  152 

Heidenhain's  haemntoxylin  stain,       .        '.;.:     ..,,;-  '.      ., .  154 

Heidenhain's  iron-lack-h<ematoxyliu,        .         ...         :  154 

Pal's  method,       ...         .         .         „,  ..; •  ...       .' ^ -  :.>.  150 

Weigert's  hsematoxylin,      :        .  ,     .       .%  '    ;.     ...^    .*. •..  155 

Hsematoxylin  and  acid  fuchsin,  double  stain,           .  ..  ...         1(>2,  103 

"      eosin,  double  stain,           -.^ .  , •.-.      .,.,,162,  103 

Hsematoxylin  and  eosin,  blood  stain,       .      ,.      ..  ,  u-.,-  •  ...  ,  ,   171 

"      Orange  G,             .        .•   ~  >    ~    •.  -{    •   •      •  164 

Hsemrin  ciystals,          .         .         .         .         .       -•'  j  •: •••''!] : '•-•'•"•-.  :'v -.»..'  ,9 

Hairr  .        .        ,'./..   .     ;  V    .   ...    .       ..    '  . ..    :, ,-  74 

Hair  follicle,        .        .        .        .        .        .'        .         .         .      .      74,  75 

Hardening  methods,           .         .         .;,./..         .     i.^-,...  128 

alcohol,  95%,         .         .         .    V.         .'.       ;,.     ',•.;'    .:v  .  '   128 

Benda's  method,           .         .        ..       '.  -   !  :..-   ^-.         .       :;.- .  130 

bichloride  of  mercury,          *        .         .         .     f*,  .'    _   .         .  128 

Flemming's  solution,           .        .  •?  '.  _v  ...  -..t.  ••.  H-.  .:,..•  I:., .. . :  130 

Hermann's  solution,             .     '    .      -.  .    _-    ..    ,    ...         .  131 

Huber's  solution,        \     ...       ,        (.    ?  .^     (    .     >.,-.;...'  131 

Miiller's  fluid,       .         .         .         .        '.,       ..'    .  .  •  »;v/'  ':   .  129 

mercuric  chloride,         .         .         .         .      :.      >  .  ,.     .        .•  128 

mercuric  chloride  and  formalin,       -  .     -.- v        .     •-.       -.,-  129 

mercuric  chloride  and  picric  acid,     -..-•,         .    ,..^.      .  129 

nitric  acid,             ".  '     .         .      ••.,*  •'.-•'       •.     •  ..         •  130 

picric  acid,             .         .     ;;v         .         .         .         .         >         .  130 

Eabl's  solution,             .         .         .        „:        .        .-      ..         .  129 

Haug's  chrom-osmium  sol.  for  decalcifying,             k     .  ;,       .  133 

Hayem's  solution,       .    .     .         .   ,      .        •.   »     .        *         .         .  173 

Heidenhain's  haematoxyUn,    '         '  ".     '   .  •  '  •*     "'&        .         .    ':  154 

iron-lack-hsematoxylin,      .         .         /  '  '  '._        .  154 

Hermann's  solution,            ,         .         .       '...-..         .  131 

Huber's  method  of  fixing  sections  to  slide  or  cover,  •      .        .  148 

**".     hardening  solution,           *   Y    *    .    "    .         .         .         .  131 

Hydrochloric  acid  for  macerating,         '     .     ' •;>.;;      .'       .         .  125 

IMPREGNATION  of  tissues,             .  '      .         .         .         .         .         .  134 

gold  chloride,  Ranvier's  method,       .....  130 

Golgi's  method,            .         .        .        .        .        .                 .  135 


INDKA.  189 

llamon  y  Cajal's  method,            .       ...   '.;•..  /•    ,;.       .         .  135 

silver  nitrate,        .  .      .  .      .  .   ..    ,     V  „  •'.; •,  .  .;*?•'  nl- •-..  .  13-1 

Injecting  tissues, 

Berlin  blue  mass,          .         .   '.."*••'".  - ..    ,  .*.••:     \     ../. ,    ......  139 

carmine  gelatin,            .•        .         .•',_    <s:i.V'V     r        •         •  ^ 

Intestine,  large,           .    i-  •;' .         .-      ,, .,,...  .... .  .,      ,.     ,    .      *.  W2 

small,            •  .  4--\  .--v  vV.^v-^iJ*-*-'    ,,.''•  91,92 

KAUYOKINESIS  in  animal  cells,            I  •    '•>•  • '••'.»   .  •*. .     •'   .      ';.  - 

"  plant  cells,               .        v        .  j    %  ••••»-, ••»-*.;*'  i 

Kidney,        .       ^k      ^ ;.  -       .•       .         j   ,'•#*•  -fJ>^-  .-  '     .        '.  103 

tubules  of,      :.     .  .        .  \ ; '^ :  --y--  •  .  •     ''.  -.      .  103 

"       human,           .         •         ..;....         .     '  ;l  • v "  :r<  104 

LARGE  intestine,          .         .,        .        .*        .         .         .      '  .  '      .  92 

Lens,             .         .        ;         .        » .'  :    .  :>      ..''   •/;'        .         .         .  116 

Lepkowski's  decalcifying  fluid,          .  '  .  .; •','-".'  .  "     .    '     '.         .  133 

Li^ainentuin  nuchfe,         '  .«/     .  "      ;        '.         .      '".         .         .  25 

Lip,     ..        ..  '      .'      . '*     l.'       '.     "  ll  'M'<:;':';';'-  *;*'•'"  '.         .  77 

Lithium  carmine,         .         .     '^        ?       \  '      ;'lF-.'''c<Jif'  '; 'r?!'  "'' .  158 

Liver,        '     'J    '   ..   [     .        V  '     ;,     •   '.  •      ,  '      .  ']    .    ''  -.    '     '.  97 

cells,         ,  •   '  v ••;  "*:       '.'\    .  '      .-'.'•     ,.;'  «  :,  ''  *'.•-  97 

of  pig,       .        .'       .      '  .  '      .  ;.  :  .V-      .        .  •     .    -  -..  97 

"       of  man,     .".       ,.         .        ':     '  \      •  '.   -.  •  ,         .        ''.  t!^}\:".  97 

Lung,            .        .        .        .    •   '.    :  ''.  .-     .-,-.'    ..  '  '  ./  .      98 

Lymph  cells,        .         .••     .         ,   •  f  .         .         .   •     \   •').••  •=>  A      69 

gland,      .    :     .  •      .     •   .  •      .  •     '.''•••     .  •      .»-    h'    ;1  68 

MAMMARY  gland,      '  .      ..        ...       ...'.:        .        .  112 

Macerating  tissues,  methods  of,         .      '•.'  ;    .      ''.'        •.?'"•  ^2^ 

alcohol,  Ranvier's,       .    .     .  '      .  '      .'    '  .'     * '.      :f.       '.  125 

caustic  potash,      .        .        .         .  ^      ,.'.*.•      .'       '.  125 

liydrochloric  acid,         .:  •  t  ...        .  '  •  .-        .        .  "      .'•     -.  •     125 

nitric  acid,             ._       .•       .        .       -.        •  •     .•        •       -•  126 

osmie  acid,             .  •      .'•      .  •      .  •      .  •    >'•      .     '-..     '   .  .     126 

Schullze's  mixture,      .    ;     .  ;      .        .  -      .  •     .  •     .        .  .    126 

sulphuric  acid,      .        .  J    ,  .      .  .      .         ,•*      .         :-.    .  .     126 

Mercuric  chloride,      .        .     .    .,      .  .*     v-i;*  x- .-.-;,,.     ..       .. :  128 

"            "           and  formalin,       .    .     ..      ..    -.    ..;  /''  .-.  129 

and  picric  acid,             .    .    ..•  .    ..       \      ..  129 

Methylene  blue,          .  .'     .        .        .      -v\    .   ,    .        .        .  161 

Methyl  green,     .        .'       ....    .....,'•<,•.«        •  161 

Methyl  violet,      .        „        .  .      .        ,V     'vV;-/-;.  ,-    ..   •;  ;.  161 

Mitosis,  animal  cells,     .     .        ..._.>..-...   .r-   i   .    ,     .  2 

plant  cells,             .   .    ^ .-.." .  ..  .     .  .     .  .  ,.V'.-,.  •«     •  1 

Miiller's  fluid,     .  .     ....      .        .        .       ,,      ,  .,        .        .  .129 

Muscle,         .        .        .        .        .        ....,.'..        .  41 

"       branched  striped,          .        .        .        .....       ..  41 

"       heart  muscle,         ........  45 

"       involuntary, ...        .     -   .  46 


m  INDEX. 

Muscle,  nerve-ending  in  striped  muscle,   •       .        .        .r)     ,  '      45 

"       striped,  voluntary,          .         ^                 ...      .        .  41,42 

NERVE  cells,         .        .        .'.      ..      »,      .  Y   .        .:    •  .1       ^  53 

"          "        of  brain  cortex,            ....        .        .  54 

"        of  cerebellum,      .        .         .    ,    *        .        .        v  54 

"          "        of  posterior  root  ganglion,           .        .        .'  •     .  .*.;  55 

"          "        of  spinal  cord,              5:) 

Nerve  fibres, .•<.••  Y  «,Y    *        •  49 

"          "        med  nl  luted,          <        .        .        f:       .        .        .  49 

"          "        non-medullated,           .        .        .  \     •  •/.'•        .  50 

Nerve  trunk,        .        .        .                 .  .     ',  ^    ..>'        .        .  50.  51 

Nitric  acid,            .               ,        .        .        .  e     ....  12(> 

"        "     for  hardening,          .        .        .  *'..'*•  \  -       •        •  1-'*0 

Nitric  and  hydrochloric  acid,             .        .-       »     •  -        »        .  l:W 

OESOPHAGUS,       ,        .        .        .        .        .        .        .        .        .  89 

il           and  cardiac  end  of  stomach,        .        .        .        ,  cS9 

Olfactory  mucous  membrane,  section,       .        .        .        .        .  119 

teased,        .        .        v  {'     .    "    *  118 

Optic  papilla,       .         .        .        .        .                 .        «'.-     v      /•  117 

Osmic  acid,          .        . „  126 

Ovary,          .        .        .        - ,        .  110 

I'AL'S  modified  Weigert's  method, 15fi 

Pancreas,    V,        ,    . 93 

Paraffin,  hard, 145 

soft,       .        .  *     «^     * ''.    .        .        .        .        ..       .  f    144 

Peptic  glands -  »  . ; ;  .  /     .,        .  90 

Picric  acid  for  hardening,          .        .        .        .        .        .        .  130 

Picro-lithiuni  carmine, 159 

Prostate,     >        .  '     i 105 

RAMON  Y  CAJAL'S  method, 135 

Removing  paraffin    from    paraffin  sections  preparatory   to 

staining,             .        .\     «; 148 

Retina,         .        .        .        •  .     *        •        .      .«  :     «     '  •-      <"  11-7 

SAFRANIN  solution,      ,        .   .     «        .        .        *     ,*   -    «        .  160 

Salivary  gland,  mucous,    .'       .       .*    .    «        -i  ••     «   -  -  .  -     4  78 

"           "       serous,      .  '',,-«    .    .  •••'*:..    «•      4        *  78 

Scalp,       '    .      >  *   w'      .      "'i,  ;'•:*,•.    *        .        „        .       v  74 

Schlemm,  canal  of,    «   ,    .    r  *        4        .'      '«'       *        .   •     .  116 

Schultze's  mixture,     .        .        *,.       *    t    «'      .;..,    « .•>     *        .  v  126 

Serial  celloidin  sections,  Weigert's  method,    «        ^       »        «  143 

Silver  nitrate,    »        .    -..«:*..        *        f        «        .   .  134 

Skin,  human,     >        *        .       V^j    •   .    *        .        .        .        .  73 

Small  intestine,           *  '•..  v.  _    V       .        .        ...  •   -    *        .  91,92 

Spermatogenesis,        .      .  .  '•.'  .i   .,   *        .        .        .        .        .  :-  109 

Spinal  cord,         .        »,      •    >    •  •     •  ,.  v»  ^  '•«>  •••'-•  -;..'.•'•      -  57 

"        "      with  anterior  and  posterior  roots,      ^        *  --*••'  57 

"        "      Golgi  stained,        .    .    *   ".    * .      i  .      .        *    tV'  53 


1KDEX.  191 

Spinal  Cord,  Weigert  stained,  .         .         .  .         .         .         .    .  57 

Spleen, »' ,     . '"';    .        .  69 

"       pulp, -•  .;••••         .        .  69 

Spreading  blood  on  cover  glasses,     .        ...        ...  169 

Staining  sections, ;•       i.       . •  ••'*  _..  lol 

Stomach,  cardiac  end,         .         .         .         .  .-..'..'•  ».'  90 

"         pyloric  end,         ....     .  .     f    .    '    .        .  90 

Sulphuric  acid  for  macerating,        <'.?       ,    .  -.         .        ..  '  ;.  126 

Suprarenal  body,         .        -.        .        ,         ...  .    'V      '.        .  104 

TASTE  buds,         .       " .,,       .        .      :  .        W'  .        •*        •        .  78 

Teichniann's  crystals,         .         .         '.        .'.  .        .      '\_  ..  9 

Tendon,       .        .        .        .        .        .        .  .        .  '• .    .        t   22,23 

Testis,          .        ...,.'...        .•.  .       .'.        .        .  109 

Thyroid  gland,     .        .       \      =.        .'     .„  .        .        .'      .  4:  99 

Tliymus  gland,    .         .         .         .«        .. /     .  .         .'     '    .        .  67 

Toison's  solution,       .        ...        ..      ^.;  .  i       '•   .    •-        -V  173 

Tonsil,          .      -,..      '.        .. '      f        .      \/./  .        .        .•       ^  67 

Tooth,           .        .        .        .        '..•     •  .     :  Vv;  .     '    .-:•  •:'. '".        .  83 

"      decalcified,       .^:..       .         .       -v:  :.   ' '   -.      -.        .  83 

"      developing,      .        .        .        *        ;     .  «  .    "••»'        .        «  84 

Trachea,       .        .       -.*      :;       ..   *v>        .  ,;::  ;,  ;    ,  ;,  >('.  98 

Triple  staining,    .        .        .        .        ...      .  '.      -'v    '  .-        •  167 

UTERUS,        .        .        *     ~  •        •        •       .»  •..'      ...        .  Ill 

VAGINA,       .      ''.••  s  ' »,       ;      \        .        .'  .     :  •     ;'.,      -  111 

Vas  deferens,      .        .v-';.        •        ...        .        .   ,     .  109 

Veins,          .        .        .v  ?1        .        .        .*    .  .     "^        .'  '  .  63 

WEIGERT'S   heematoxylin,          .        .        .  . .' -  ^W     •        •  155 

White  blood  cells,       .        .        .        .        .  r        .       v        .  6 


14  DAY  USE 

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^181952 

JAN  -8:1962 

